Methods and compositions for treatment of a genetic condition

ABSTRACT

Methods and compositions for genetic alteration of cells are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of U.S. ProvisionalApplication No. 61/823,689, filed May 15, 2013, the disclosure of whichis hereby incorporated by reference in its entirety.

TECHNICAL FIELD

The present disclosure is in the field of genome engineering.

BACKGROUND

Gene therapy holds enormous potential for a new era in human medicine.These methodologies will allow treatment for conditions that heretoforehave not been addressable by standard medical practice. One area that isespecially promising is the ability to genetically engineer a cell tocause that cell to express a product that has not previously beenproduced in that cell. Examples of uses of this technology include theinsertion of a transgene encoding a novel therapeutic protein, insertionof a coding sequence encoding a protein that is lacking in the cell orin the individual, insertion of a wild type gene in a cell containing amutated gene sequence, and insertion of a sequence that encodes astructural nucleic acid such as a microRNA or siRNA.

To meet the challenge of increasing global demand for food production,many effective approaches to improving agricultural productivity (e.g.enhanced yield or engineered pest resistance) rely on either mutationbreeding or introduction of novel genes into the genomes of crop speciesby transformation. Both processes are inherently non-specific andrelatively inefficient. For example, conventional plant transformationmethods deliver exogenous DNA that integrates into the genome at randomlocations. The random nature of these methods makes it necessary togenerate and screen hundreds of unique random-integration events perconstruct in order to identify and isolate transgenic lines withdesirable attributes. Moreover, conventional transformation methodscreate several challenges for transgene evaluation including: (a)difficulty for predicting whether pleiotropic effects due to unintendedgenome disruption have occurred; and (b) difficulty for comparing theimpact of different regulatory elements and transgene designs within asingle transgene candidate, because such comparisons are complicated byrandom integration into the genome. As a result, conventional planttrait engineering is a laborious and cost intensive process with a lowprobability of success.

Precision gene modification overcomes the logistical challenges ofconventional practices in plant systems, and as such has been alongstanding but elusive goal in both basic plant biology research andagricultural biotechnology. However, with the exception of “genetargeting” via positive-negative drug selection in rice or the use ofpre-engineered restriction sites, targeted genome modification in allplant species, both model and crop, has until recently proven verydifficult. Terada et al. (2002) Nat Biotechnol 20(10):1030; Terada etal. (2007) Plant Physiol 144(2):846; D'Halluin et al. (2008) PlantBiotechnology J. 6(1):93.

Transgene (or trait) stacking has great potential for production ofplants, but has proven difficult. See, e.g., Halpin (2005) PlantBiotechnology Journal 3:141-155. In addition, polyploidy, where theorganism has two or more duplicated (autoploidy) or related (alloploid)paired sets of chromosomes, occurs more often in plant species than inanimals. For example, wheat has lines that are diploid (two sets ofchromosomes), tetraploid (four sets of chromosomes) and hexaploid (sixsets of chromosomes). In addition, many agriculturally important plantsof the genus Brassica are also allotetraploids.

Transgenes can be delivered to a cell by a variety of ways, such thatthe transgene becomes integrated into the cell's own genome and ismaintained there. In recent years, a strategy for transgene integrationhas been developed that uses cleavage with site-specific nucleases fortargeted insertion into a chosen genomic locus (see, e.g., co-owned U.S.Pat. No. 7,888,121). Nucleases specific for targeted genes can beutilized such that the transgene construct is inserted by eitherhomology directed repair (HDR) or by end capture during non-homologousend joining (NHEJ) driven processes. Targeted loci include “safe harbor”loci such as the AAVS1, HPRT and CCR5 genes in human cells, and Rosa26in murine cells (see, e.g., U.S. Pat. Nos. 7,888,121; 7,972,854;7,914,796; 7,951,925; 8,110,379; 8,409,861; 8,586,526; U.S. PatentPublications 20030232410; 20050208489; 20050026157; 20060063231;20080159996; 201000218264; 20120017290; 20110265198; 20130137104;20130122591; 20130177983 and 20130177960) and the Zp15 locus in plants(see U.S. Pat. No. 8,329,986). Nuclease-mediated integration offers theprospect of improved transgene expression, increased safety andexpressional durability, as compared to classic integration approachesthat rely on random integration of the transgene, since it allows exacttransgene positioning for a minimal risk of gene silencing or activationof nearby oncogenes.

Genome engineering can also include the knocking out of genes inaddition to insertion methods described above. In the absence of a donornucleic acid, a cell with a cleaved genome will resort to the errorprone NHEJ pathway to heal the break. This process often adds or deletesnucleotides during the repair process (“indels”) which may lead to theintroduction of missense or non-sense mutations at the target site. Forexample, CCR5-specific zinc finger nucleases are being used in PhaseI/II trials to create a non-functional CCR5 receptor, and thus preventHIV infection (see U.S. Pat. No. 7,951,925).

Targeted nuclease-mediated genome cleavage at a desired location can beobtained by the use of an engineered nuclease. For example, adouble-strand break (DSB) for can be created by a site-specific nucleasesuch as a zinc-finger nuclease (ZFN) or TAL effector domain nuclease(TALEN). See, for example, Urnov et al. (2010) Nature 435(7042):646-51;U.S. Pat. Nos. 8,586,526; 6,534,261; 6,599,692; 6,503,717; 6,689,558;7,067,317; 7,262,054, the disclosures of which are incorporated byreference in their entireties for all purposes.

Another nuclease system involves the use of a so-called acquiredimmunity system found in bacteria and archaea known as the CRISPR/Cassystem. CRISPR/Cas systems are found in 40% of bacteria and 90% ofarchaea and differ in the complexities of their systems. See, e.g., U.S.Pat. No. 8,697,359. The CRISPR loci (clustered regularly interspacedshort palindromic repeat) is a region within the organism's genome whereshort segments of foreign DNA are integrated between short repeatpalindromic sequences. These loci are transcribed and the RNAtranscripts (“pre-crRNA”) are processed into short CRISPR RNAs (crRNAs).There are three types of CRISPR/Cas systems which all incorporate theseRNAs and proteins known as “Cas” proteins (CRISPR associated). Types Iand III both have Cas endonucleases that process the pre-crRNAs, that,when fully processed into crRNAs, assemble a multi-Cas protein complexthat is capable of cleaving nucleic acids that are complementary to thecrRNA.

In type II systems, crRNAs are produced using a different mechanismwhere a trans-activating RNA (tracrRNA) complementary to repeatsequences in the pre-crRNA, triggers processing by a doublestrand-specific RNase III in the presence of the Cas9 protein. Cas9 isthen able to cleave a target DNA that is complementary to the maturecrRNA however cleavage by Cas 9 is dependent both upon base-pairingbetween the crRNA and the target DNA, and on the presence of a shortmotif in the crRNA referred to as the PAM sequence (protospacer adjacentmotif) (see Qi et al (2013) Cell 152:1173). In addition, the tracrRNAmust also be present as it base pairs with the crRNA at its 3′ end, andthis association triggers Cas9 activity.

The Cas9 protein has at least two nuclease domains: one nuclease domainis similar to a HNH endonuclease, while the other resembles a Ruvendonuclease domain. The HNH-type domain appears to be responsible forcleaving the DNA strand that is complementary to the crRNA while the Ruvdomain cleaves the non-complementary strand.

The requirement of the crRNA-tracrRNA complex can be avoided by use ofan engineered “single-guide RNA” (sgRNA) that comprises the hairpinnormally formed by the annealing of the crRNA and the tracrRNA (seeJinek et al (2012) Science 337:816 and Cong et al (2013)Sciencexpress/10.1126/science.1231143). In S. pyrogenes, the engineeredtracrRNA:crRNA fusion, or the sgRNA, guides Cas9 to cleave the targetDNA when a double strand RNA:DNA heterodimer forms between the Casassociated RNAs and the target DNA. This system comprising the Cas9protein and an engineered sgRNA containing a PAM sequence has been usedfor RNA guided genome editing (see Ramalingam ibid) and has been usefulfor zebrafish embryo genomic editing in vivo (see Hwang et al (2013)Nature Biotechnology 31 (3):227) with editing efficiencies similar toZFNs and TALENs.

Thus, there remains a need for systems for genomic editing, includingfor treatment and/or prevention of diseases and for agricultural uses.

SUMMARY

Disclosed herein are methods and compositions for genetic modification,for example for the treatment of a disease or for production ofgenetically modified plants. Genome editing is used to correct anaberrant gene, insert a wild type gene, or change the expression of anendogenous gene. One or more endogenous genes can be targeted forgenomic editing, including any mammalian or plant gene(s). By way ofnon-limiting example, a wild-type gene encoding β globin may be insertedinto the genome of a cell to treat a hemoglobinopathy caused by faulty βglobin. In some instances, the wild type gene may be inserted into asafe harbor locus or at a locus known to be highly expressed in a tissueof interest such as the β globin locus in erythroid cells. Anotherapproach disclosed here involves the use of gene correction where afaulty endogenous gene is targeted and the mutant sequence replacedusing an engineered donor (e.g., the mutant is replaced with afunctional sequence). Alternately, one or more regulatory genes involvedin the modulation of another gene may be altered, for example aregulatory gene involved in repression of a gene may be altered orknocked out such that the normally repressed gene is now expressed ornot expressed, or the regulatory binding site upstream of the a gene orin other areas of the locus are altered so that the regulators are notable to interact properly with the DNA at the gene locus and regulategene expression. Another approach further involves the use of modifiedstem cells (e.g., hematopoietic stem cell or red blood cell (RBC)precursor), which can then be used to engraft into a patient, fortreatment of a hemoglobinopathy or other disease (e.g., geneticdisease).

In one aspect, described herein is a CRISPR/Cas system that binds totarget site in a region of interest in an endogenous gene (e.g., anendogenous or safe harbor gene, or a regulatory gene or its DNA target)in a genome, wherein the CRISPR/Cas system comprises one or moreengineered single guide RNAs that recognize the target gene and afunctional domain (e.g., a transcriptional regulatory domain and/or anuclease domain).

The CRISPR/Cas system as described herein may bind to and/or cleave theregion of interest in a coding or non-coding region within or adjacentto the gene, such as, for example, a leader sequence, trailer sequenceor intron, or within a non-transcribed region, either upstream ordownstream of the coding region. In certain embodiments, the CRISPR/Casbinds to and/or cleaves a gene. In other embodiments, the CRISPR/Casbinds to and/or cleaves a safe-harbor gene, for example a CCR5 gene, aPPP1R12C (also known as AAVS1) gene, or a Rosa gene in mammalian cells,and the Zp15 locus in plants. PPP1R12C has 22 exons in its codingsequence, and an especially preferred location for targeting is withinintron 1 (i.e. at or near chr19:55624164-55624759), enabling theinsertion of a promoter-less transgene. In addition, to aid in selectionin mammalian systems, the HPRT locus may be used (see U.S. applicationSer. Nos. 13/660,821 and 13/660,843). In some embodiments, theCRISPR/Cas binds to and cleaves at regulatory elements. In anotheraspect, described herein are compositions comprising one or more of theCRISPR/Cas nucleases as described herein.

In one aspect, the CRISPR/Cas system as described may bind to and/ormodulate expression of a gene of interest. In one embodiment, theCRISPR/Cas system binds to a DNA sequence and prevents binding of otherregulatory factors. In another embodiment, the binding of a CRISPR/Cassystem fusion protein may modulate (i.e. induce or repress) expressionof a target DNA.

In another aspect, described is a polynucleotide encoding one or moreCRISPR/Cas system described herein. The polynucleotide may be, forexample, mRNA. In some aspects, the mRNA may be chemically modified (Seee.g. Kormann et al, (2011) Nature Biotechnology 29(2):154-157).

In another aspect, described is a CRISPR/Cas system expression vectorcomprising a polynucleotide encoding one or more CRISPR/Cas componentsdescribed herein, operably linked to a promoter. In one embodiment, theexpression vector is a viral vector. In another embodiment, theexpression vector is a DNA minicircle.

In one aspect, described herein is a Cas protein that is used to cleavea target DNA.

In another aspect, described herein is a method of modifying anendogenous gene (e.g., modulating expression of the endogenous gene),the method comprising administering to the cell a first nucleic acidmolecule comprising a single guide RNA that recognizes a target site inthe endogenous gene and a second nucleic acid molecule that encodes afunctional domain, wherein the functional domain associates with thesingle guide RNA on the target site, thereby modifying the endogenousgene. The first and second nucleic acids may be on the same or differentvectors. In certain embodiments, the endogenous gene is selected fromthe group consisting of a mammalian β globin gene (HBB), a gamma globingene (HBG1), a B-cell lymphoma/leukemia 11A (BCL11A) gene, aKruppel-like factor 1 (KLF1) gene, a CCR5 gene, a CXCR4 gene, a PPP1R12C(AAVS1) gene, an hypoxanthine phosphoribosyltransferase (HPRT) gene, analbumin gene, a Factor VIII gene, a Factor IXgene, a Leucine-rich repeatkinase 2 (LRRK2) gene, a Hungtingin (Htt) gene, a rhodopsin (RHO) gene,a Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene, asurfactant protein B gene (SFTPB), a T-cell receptor alpha (TRAC) gene,a T-cell receptor beta (TRBC) gene, a programmed cell death 1 (PD1)gene, a Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4) gene, an humanleukocyte antigen (HLA) A gene, an HLA B gene, an HLA C gene, an HLA-DPAgene, an HLA-DQ gene, an HLA-DRA gene, a LMP7 gene, a Transporterassociated with Antigen Processing (TAP) 1 gene, a TAP2 gene, a tapasingene (TAPBP), a class II major histocompatibility complex transactivator(CIITA) gene, a dystrophin gene (DMD), a glucocorticoid receptor gene(GR), an IL2RG gene, an RFX5 gene, a FAD2 gene, a FAD3 gene, a ZP15gene, a KASII gene, a MDH gene, and/or an EPSPS gene.

In any of the methods and compositions described herein, the cell can beany eukaryotic cell(s), for example a plant cell or a mammalian cell orcell line, including COS, CHO (e.g., CHO-S, CHO-K1, CHO-DG44,CHO-DUXB11, CHO-DUKX, CHOK1SV), VERO, MDCK, WI38, V79, B14AF28-G3, BHK,HaK, NS0, SP2/0-Ag14, HeLa, HEK293 (e.g., HEK293-F, HEK293-H, HEK293-T),and perC6 cells as well as insect cells such as Spodopterafugiperda(Sf), or fungal cells such as Saccharomyces, Pichia andSchizosaccharomyces. In certain embodiments, the cell line is a CHO,MDCK or HEK293 cell line. Primary cells can also be edited as describedherein, including but not limited to fibroblasts, blood cells (e.g., redblood cells, white blood cells), liver cells, kidney cells, neuralcells, and the like. Suitable cells also include stem cells such as, byway of example, embryonic stem cells, induced pluripotent stem cells(iPSCs), hematopoietic stem cells, neuronal stem cells and mesenchymalstem cells. In other aspects, genetically modified blood cell precursors(hematopoietic stem cells known as “HSCs”) are given in a bone marrowtransplant and the HSCs differentiate and mature in vivo.

In other aspects, genetically modified RBC precursors and/orhematopoietic stem cells (HSCs) are given in a bone marrow transplantand the RBCs differentiate and mature in vivo. In some embodiments, theHSCs are isolated from the peripheral blood following G-CSF-inducedmobilization, and in others, the cells are isolated from human bonemarrow or umbilical cord blood. In some aspects, the HSCs are edited bytreatment with a nuclease designed to knock out a specific gene orregulatory sequence. In other aspects, the HSCs are modified with anengineered nuclease and a donor nucleic acid such that a wild type geneis inserted and expressed and/or an endogenous aberrant gene iscorrected. In some embodiments, an engineered gene is inserted andexpressed. In some embodiments, the modified HSCs are administered tothe patient following mild myeloablative pre-conditioning. In otheraspects, the HSCs are administered after full myeloablation such thatfollowing engraftment, a majority of the hematopoietic cells are derivedfrom the newly engrafted modified HSC population.

In one aspect, the invention comprises mutated Cas nucleases. In someembodiments, the mutant Cas nucleases are Cas9 nucleases, and havealtered functionality. In some embodiments, the Cas9 protein is mutatedin the HNH domain, rendering it unable to cleave the DNA strand that iscomplementary to the crRNA. In other embodiments, the Cas9 is mutated inthe Rvu domain, making it incapable of cleaving the non-complimentaryDNA strand. These mutations can result in the creation of Cas9 nickases.In some embodiments, two Cas nickases are used with two separate crRNAsto target a DNA, which results in two nicks in the target DNA at aspecified distance apart. In other embodiments, both the HNH and Rvuendonuclease domains are altered to render a Cas9 protein which isunable to cleave a target DNA.

In another aspect, the methods and compositions of the inventioncomprise truncations of the Cas9 protein. In one embodiment, the Cas9protein is truncated such that one or more of the Cas9 functionaldomains are removed. In one embodiment, the removal of part or one ofthe nuclease domains renders the Cas nuclease a nickase. In oneembodiment, the Cas9 comprises only the domain responsible forinteraction with the crRNA or sgRNA and the target DNA.

In still further aspects, the methods and compositions of the inventionalso comprise fusion proteins wherein the Cas9 protein, or truncationthereof, is fused to a functional domain. In some aspects, thefunctional domain is an activation or a repression domain. In otheraspects, the functional domain is a nuclease domain. In someembodiments, the nuclease domain is a FokI endonuclease domain (e.g.Tsai (2014) Nature Biotech doi:10.1038/nbt.2908). In some embodiments,the FokI domain comprises mutations in the dimerization domain.

In another aspect, described herein is a method for inserting a sequenceinto an endogenous gene (e.g., safe harbor gene) in a cell (e.g. stemcell), the method comprising cleaving the endogenous gene using one ormore nucleases and inserting a sequence into the cleavage site. Incertain embodiments, a genomic sequence in any target gene is replaced,for example using a CRISPR/Cas system (or vector encoding saidCRIPSR/Cas system) as described herein and a “donor” sequence (alsoknown as a “transgene”) that is inserted into the gene followingtargeted cleavage. The donor sequence may be present in the CRISPR/Casvector, present in a separate vector (e.g., Ad, AAV, DNA minicircle orLV vector) or, alternatively, may be introduced into the cell using adifferent nucleic acid delivery mechanism. For example, mRNA encodingthe Cas nuclease protein may be introduced into a cell with the desiredsgRNA by electroporation. Such insertion of a donor nucleotide sequenceinto the target locus (e.g., safe-harbor gene) results in the expressionof the transgene under control of the target locus's genetic controlelements. In some embodiments, the transgene encodes a non-coding RNA(e.g. an shRNA). Expression of the transgene prior to stem celldifferentiation will result in a derivative cell containing thenon-coding RNA of interest.

In some embodiments, the methods and compositions of the invention areperformed in and/or comprise plant cells. In some aspects, the plantcells are meristematic cells. In some embodiments, the plant cellscomprise a nuclease of the invention. In other embodiments, the plantcells additionally comprise a transgene. In yet another aspect,described herein is a method for introducing one or more exogenoussequence into the genome of a plant cell, the method comprising thesteps of: (a) contacting the cell with the one or more exogenoussequences (donor vector, transgene or GOI); and (b) expressing one ormore nucleases (e.g., CRISPR/Cas system) as described herein in thecell, wherein the one or more nucleases cleave chromosomal DNA; suchthat cleavage of chromosomal DNA in step (b) stimulates incorporation ofthe donor vector into the genome by homologous recombination. Multipletransgenes may be integrated simultaneously (in parallel) or the stepsmay be repeated for sequential addition of transgenes (transgenestacking).

In other embodiments, the transgene comprises a functional protein, forexample a globin (e.g., wild type beta and/or wild type gamma) protein.In other embodiments, the transgene encodes an engineered gene forproduction of a novel protein with desirable qualities. In someembodiments, insertion of the transgene of interest into an endogenousgene, results in expression of an intact exogenous protein sequence andlacks any sequences encoded by the endogenous gene. In otherembodiments, the expressed exogenous protein is a fusion protein andcomprises amino acids encoded by the transgene and by the endogenousgene (e.g., from the endogenous target locus). When present, endogenoussequences may be present on the amino (N)-terminal portion of theexogenous protein and/or the carboxy (C)-terminal portion of theexogenous protein. In some aspects, the safe harbor is selected from theAAVS1, Rosa, HPRT, Zp15 or CCR5 locus (see U.S. Patent Publications Nos.20080299580; 20080159996; and 201000218264 and U.S. application Ser.Nos. 13/660,821 and 13/660,843 and U.S. Pat. No. 8,329,986).

In yet another aspect, provided herein are genomically modified celllines and/or transgenic organisms such as animal models (systems) Insome embodiments, the transgenic cell and/or organism (e.g., animal)includes a transgene that encodes a human gene. In some instances, thetransgenic animal comprises a knock out at the endogenous locuscorresponding to exogenous transgene, thereby allowing the developmentof an in vivo system where the human protein may be studied inisolation. Such transgenic models may be used for screening purposes toidentify small molecules or large biomolecules or other entities whichmay interact with or modify the human protein of interest. In someembodiments, the cell lines of the invention are used in cell-basedassays for activity screens in pharmaceutical development, while inother embodiments, the cell lines are used in diagnostic assays. In someaspects, the transgene is integrated into the selected locus (e.g.,safe-harbor) into a stem cell (e.g., an embryonic stem cell, an inducedpluripotent stem cell, a hematopoietic stem cell, etc.) or animal embryoobtained by any of the methods described herein, and then the embryo isimplanted such that a live animal is born. The animal is then raised tosexual maturity and allowed to produce offspring wherein at least someof the offspring comprise edited endogenous gene sequence or theintegrated transgene.

In a still further aspect, a cell (e.g., plant or mammalian cell)obtained according to any of the methods described herein is alsoprovided.

In another aspect, provided herein is an organism (e.g., plant oranimal) comprising a cell (e.g., plant or animal) as described herein.

In another aspect, provided herein is a seed from a plant comprising theplant cell that is obtained as described herein.

In another aspect, provided herein is fruit obtained from a plantcomprising plant cell obtained as described herein.

In any of the compositions (cells or plants) or methods describedherein, the plant cell can comprise a monocotyledonous or dicotyledonousplant cell. In certain embodiments, the plant cell is a crop plant, forexample, wheat, tomato (or other fruit crop), potato, maize, soy,alfalfa, etc.

In a still further aspect, provided herein is a method for site-specificintegration of a nucleic acid sequence into an endogenous locus (e.g.,safe harbor gene) of a chromosome, for example into the chromosome of anembryo. In certain embodiments, the method comprises: (a) injecting anembryo with (i) at least one DNA vector, wherein the DNA vectorcomprises an upstream sequence and a downstream sequence flanking thenucleic acid sequence to be integrated, and (ii) at least one RNAmolecule encoding a CRISPR/Cas system nuclease that recognizes the siteof integration in the target locus (e.g., globin or safe harbor locus),and (b) culturing the embryo to allow expression of the CRISPR/Cassystem nuclease, wherein a double stranded break introduced into thesite of integration by the CRISPR/Cas system nuclease is repaired, viahomologous recombination with the DNA vector, so as to integrate thenucleic acid sequence into the chromosome.

In any of the methods described herein, the polynucleotide encoding theCRISPR/Cas system can comprise DNA, RNA or combinations thereof. Incertain embodiments, the polynucleotide comprises a plasmid. In otherembodiments, the polynucleotide encoding the nuclease comprises mRNA.

A kit, comprising a CRISPR/Cas system of the invention, is alsoprovided. The kit may comprise nucleic acids encoding a CRISPR/Cassystem, (e.g. RNA molecules or CRISPR/Cas system encoding genescontained in a suitable expression vector), or aliquots of the nucleaseproteins, donor molecules, suitable host cell lines, instructions forperforming the methods of the invention, and the like.

These and other aspects will be readily apparent to the skilled artisanin light of disclosure as a whole.

DETAILED DESCRIPTION

Disclosed herein are methods and compositions for genome engineering,including genome engineering to study and treat a disease or for thecreation of a plant with desirable traits. The invention describesgenomic editing of any target cell such that there is a favorable changein the expression of one or more genes, which in turn results intreatment of a disease in a subject in need thereof or the production ofa desirable plant. Non-limiting examples of diseases include geneticdiseases (e.g., hemoglobinopathies, hemophilia, lysosomal storagediseases, cystic fibrosis etc.), infectious diseases (e.g., HIV),neurological diseases (e.g., PD, HD, etc.), cancers, and the like.Additionally, delivery of altered stem cells in a transplant altered toexpress a desired protein product can be similarly beneficial in adisease. Also described are cell lines and organisms (e.g., plants oranimals) with altered gene expression. Described below are genes to betargeted by the CRISPR/Cas system using the sgRNAs of the invention.Mammalian gene locations as described are relative to the UCSC GenomeBrower created by the Genome Bioinformatics Group of UC Santa Cruz,software copyright the Regents of the University of California. Humangenomic coordinates are provided in the GRCh37/hg19 assembly of thehuman genome, and correspond to numbers on a double stranded DNA. Thus,any position described by a genomic coordinate corresponds to either the(+) or Watson strand, or may specify its corresponding (−) or Crickstrand.

Exemplary targets include genes involved in hemoglobinopathies.Hemoglobin is a heterotetramer comprising two α-like globin chains andtwo β-like globin chains and 4 heme groups. In adults the α2β2 tetrameris referred to as Hemoglobin A (HbA) or adult hemoglobin. Typically, thealpha and beta globin chains are synthesized in an approximate 1:1 ratioand this ratio seems to be critical in terms of hemoglobin and RBCstabilization. In fact, in some cases where one type of the globin genesis inadequately expressed (see below), reducing expression (e.g. using aspecific siRNA) of the other type of globin, restoring this 1:1 ratio,alleviates some aspects of the mutant cellular phenotype (see Voon et al(2008) Haematologica 93(8):1288). In a developing fetus, a differentform of hemoglobin, fetal hemoglobin (HbF) is produced which has ahigher binding affinity for oxygen than Hemoglobin A such that oxygencan be delivered to the baby's system via the mother's blood stream.Fetal hemoglobin also contains two a globin chains, but in place of theadult β-globin chains, it has two fetal γ-globin chains (α2γ2). Atapproximately 30 weeks of gestation, the synthesis of γ-globin in thefetus starts to drop while the production of β-globin increases. Byapproximately 10 months of age, the newborn's hemoglobin is nearly allα2β2 although some HbF persists into adulthood (approximately 1-3% oftotal hemoglobin). The regulation of the switch from production of γ toβ is quite complex, and primarily involves an expressionaldown-regulation of γ-globin with a simultaneous up-regulation ofβ-globin expression.

Genetic defects in the sequences encoding the hemoglobin chains can beresponsible for a number of diseases known as hemoglobinopathies,including sickle cell anemia and thalassemias. In the majority ofpatients with hemoglobinopathies, the genes encoding γ-globin remainpresent, but expression is relatively low due to normal gene repressionoccurring around parturition as described above.

It is estimated that 1 in 5000 people in the U.S. have sickle celldisease (SCD), mostly in people of sub-Saharan Africa descent. Thereappears to be a benefit of sickle cell heterozygosity for protectionagainst malaria, so this trait may have been selected for over time,such that it is estimated that in sub-Saharan Africa, one third of thepopulation has the sickle cell trait. Sickle cell disease is caused by amutation in the β-globin gene in which valine is substituted forglutamic acid at amino acid #6 (a GAG to GTG at the DNA level), wherethe resultant hemoglobin is referred to as “hemoblobin S” or “HbS.”Under lower oxygen conditions, the deoxy form of HbS exposes ahydrophobic patch on the protein between the E and F helices. Thehydrophobic residues of the valine at position 6 of the beta chain inhemoglobin are able to associate with the hydrophobic patch, causing HbSmolecules to aggregate and form fibrous precipitates. These aggregatesin turn cause the abnormality or ‘sickling’ of the RBCs, resulting in aloss of flexibility of the cells. The sickling RBCs are no longer ableto squeeze into the capillary beds and can result in vaso-occlusivecrisis in sickle cell patients. In addition, sickled RBCs are morefragile than normal RBCs, and tend towards hemolysis, eventually leadingto anemia in the patient.

Thalassemias are also diseases relating to hemoglobin and typicallyinvolve a reduced expression of globin chains. This can occur throughmutations in the regulatory regions of the genes or from a mutation in aglobin coding sequence that results in reduced expression. Alphathalassemias are associated with people of Western Africa and SouthAsian descent, and may confer malarial resistance. Beta thalassemia isassociated with people of Mediterranean descent, typically from Greeceand the coastal areas of Turkey and Italy. Treatment of thalassemiasusually involves blood transfusions and iron chelation therapy. Bonemarrow transplants are also being used for treatment of people withsevere thalassemias if an appropriate donor can be identified, but thisprocedure can have significant risks.

Correction of the human HBB gene that encodes beta globin can beaccomplished with the CRISPR/Cas system of the invention. Preferredlocations of cleavage include targeting the HBB gene sequence (i.e. ator near chr11:5246696-5248301). Especially preferred is to targetregions of HBB in a HBS allele to achieve gene correction of a sickleallele. Especially preferred for use of a S. pyogenes Cas9 system istargeting sequences where the PAM site is at or near positions onchromosome 11: 5248110, 5248106, 5248100, 5248090, 5248122, 5248112 forcorrection of a sickle allele. For gene correction of a beta-globinallele associated with a beta thalessemia there are many potentialtargets. One well known mutation associated with beta thalassemia is theso-called IVS1.1 mutation, where a preferred targeting region would bearound nucleotides 1093-1192 of the HBB gene sequence. Most preferredare targeting sequences where the PAM site is at or near positions onchromosome 11: 5248170-5248171, 5248168-5248169, 5248167-5248168,5248164-5248165, 5248163-5248164, 5248155-5248156 and 5248147-5248148.

One approach for the treatment of both SCD and beta thalassemias thathas been proposed is to increase the expression of γ-globin with the aimto have HbF functionally replace the aberrant adult hemoglobin. Asmentioned above, treatment of SCD patients with hydroxyurea is thoughtto be successful in part due to its effect on increasing γ-globinexpression. The first group of compounds discovered to affect HbFreactivation activity were cytotoxic drugs. The ability to cause de novosynthesis of gamma-globin by pharmacological manipulation was firstshown using 5-azacytidine in experimental animals (DeSimone (1982) ProcNatl Acad Sci USA 79(14):4428-31). Subsequent studies confirmed theability of 5-azacytidine to increase HbF in patients with β-thalassemiaand sickle cell disease (Ley, et al. (1982) N. Engl. J. Medicine, 307:1469-1475, and Ley, et al., (1983) Blood 62: 370-380). In addition,short chain fatty acids (e.g. butyrate and derivatives) have been shownin experimental systems to increase HbF (Constantoulakis et al. (1988)Blood 72(6):1961-1967). There is a segment of the human population witha condition known as ‘Hereditary Persistence of Fetal Hemoglobin’ (HPFH)where elevated amounts of HbF persist in adulthood (10-40% in HPFHheterozygotes (see Thein et al. (2009) Hum. Mol. Genet 18 (R2):R216-R223)). This is a rare condition, but in the absence of anyassociated beta globin abnormalities, is not associated with anysignificant clinical manifestations, even when 100% of the individual'shemoglobin is HbF. When individuals that have a beta thalassemia alsohave co-incident HPFH, the expression of HbF can lessen the severity ofthe disease. Further, the severity of the natural course of sickle celldisease can vary significantly from patient to patient, and thisvariability, in part, can be traced to the fact that some individualswith milder disease express higher levels of HbF.

One approach to increase the expression of HbF involves identificationof genes whose products play a role in the regulation of γ-globinexpression. One such gene is BCL11A, first identified because of itsrole in lymphocyte development. BCL11A encodes a zinc finger proteinthat is thought to be involved in the stage specific regulation ofγ-globin expression. BCL11A is expressed in adult erythroid precursorcells and down-regulation of its expression leads to an increase inγ-globin expression. See, Sankaran et al (2008) Science 322 p. 1839. Theprotein appears to interact with the β-globin locus to alter itsconfiguration and thus its expression at different developmental stages.In addition, another regulatory protein KLF1 (encoded atchr19:12995237-12998017), appears to be involved in regulation ofγ-globin expression. It has been found that KLF1 levels are directlyproportional to BCL11A levels, and both are inversely proportional toγ-globin levels in a Maltese family with persistent expression of HbFthat carries a heterozygous mutation of the KLF1 gene (Borg et al,(2011) Haematologica 96(5): 635-638). The KLF1 gene product appears tobind directly to the BCL11A gene in vivo, and thus may be responsiblefor its upregulation (see Borg et al (2010) Nat Genet 42(9):801-805;Bieker (2010) Nat Genet 42(9): 733-734; Zhou et al. (2010) Nat Genet42(9):742-744). Thus, if KLF1 stimulates BCL11A expression, the actionof BCL11A will result in the suppression of γ-globin and HbF production.Use of an inhibitory RNA targeted to the BCL11A gene has been proposed(see, e.g., U.S. Patent Publication No. 20110182867) but this technologyhas several potential drawbacks, namely that complete knock down may notbe achieved, delivery of such RNAs may be problematic and the RNAs mustbe present continuously, requiring multiple treatments for life. Thus,the methods and compositions of the invention may be used to treat SCDand hemoglobinopathies with a CRISPR/Cas system where the single guideRNA comprises sequences to target the KLF1 or BCL11a genes. Knock out ofthe KLF1 gene through double strand cleavage may be accomplished throughthe CRIPSR/Cas system of the invention. Preferred sites for targetingare in exon 1, exon 2 and exon 3 to give the desired knock out of thegene. Especially preferred are the regions on chromosome 19 at or near12994239-12999017, 12997868-12998017, and 12996131-12996956. Especiallypreferred is targeting the region of chromosome 19 at or near12997877-12997912. Non-limiting examples of sequences suitable fortargeting are shown in Table 1.

Knock out of the BCL11A gene through double strand cleavage may beaccomplished using a CRISPR/Cas system and the sgRNAs of the invention.Preferred is to target the BCL11A gene (chr2:60684329-60780633). Alsopreferred is to cleave the BCL11A gene in an exonic sequence (e.g. at ornear chromosome 2: 60780351-60780633 (exon 1), 60773106-60773435,especially 60773362-60773400 (exon 2), 60695867-60695968 (exon 3),60687817-60689559 (exon 4), 60678302-60679801 (exon 5)). Especiallypreferred is targeting the enhancer sequence in the BCL11A gene (seeBauer et al (2013), Science 342 (6155):253-7). Thus, targeting sequencesat or near chromosome 2 60725317-60725682, 60722125-60722677 and60718031-60718382 is especially preferred.

Another approach to alter the expression of gamma globin is to targetthe regulatory sites on the gene encoding gamma globin (HBG1, located onchromosome 11: 5268501-5272087). Preferred is to target thetranscriptional start site at or near chromosome 11:5271086-5271087,Especially preferred is to target the so-called “HPFH” region, knownbecause of patients with hereditary persistence of fetal hemoglobin (seeThein et al (2009) Hum. Mol. Genet 18 (R2): R216-R223). Thus, targetingsequences at or near chromosome 11:5272286-5272290, 5272263-5272263 and5272198-5272205 is especially preferred.

Alpha thalassemias are also prevalent in the human population,especially in Asia and some type of alpha globin aberrancy is thought tobe the commonest genetic disorder in humans. In the tropical andsubtropical areas of the world, alpha globin disorder is found in 80-90%of the population (see Harteveld and Higgs (2010) Orphanet Journal ofRare Diseases 5:13).

Humans carry 2 copies of the alpha globin gene in tandem (α1 and α2) onchromosome 16, so in a normal diploid cell there are 4 copies alltogether. The α2 gene normally accounts for 2-3 times more a-globin mRNAthan the al gene. The tandem organization of these two genes may beassociated with the high prevalence of large deletions in alpha globingenes in alpha thalessemia patients, where generally the number of alphaglobin genes that are non-functional relates directly to the severity ofany alpha thalessemia (see Chui et al (2003) Blood 101(3):791). Deletionof one copy seems to be fairly common (30% of African Americans and60-80% of people living in Saudi Arabia, India, and Thailand), and isgenerally not evident in the individual unless genetic testing is done.Deletion of two copies, whether on the same chromosome (cis) or one fromeach chromosome (trans), may cause the afflicted person to have mildanemia. When three α-globin genes are deleted, such that the individualhas only one functioning α-globin gene, moderate anemia is found, butmore importantly, the crucial a globin to β globin ratio is disrupted.β4 tetramers, comprising four beta-globin chains, are often observed inpatients with only one functional alpha-globin gene, an condition knownas HbH. The β4 tetramers are able to bind oxygen but do not release itinto the periphery, causing what is known as Hb H disease. Individualswith HbH disease have RBCs with shortened half-lives and which undergohemolysis easily, leading to increased anemia. Loss of all four a-globingenes is usually fatal in utero. Thus, the methods and compositions ofthe invention may be used to treat thalassemias with a CRISPR/Cas systemwhere the single guide RNA comprises sequences to target the regulatoryregion of the alpha globin gene. Non-limiting examples of sequencessuitable for targeting are shown in Table 1.

Other exemplary targets include genes encoding receptors, for exampleviral receptors. When HIV infects human T cells, it relies onassociation with the T cell receptor CD4 and one of two co-receptors,the chemokine receptor CCR5 or CXCR4, to gain entry into the cell.Natural CCR5 variants (“CCR5-delta 32”) in the human population wereidentified who appear to be resistant to HIV infection, especially inthe homozygous state. Thus, to prevent HIV from infecting T cells, andultimately leading to T cell death and decreased immune function in theHIV infected patient, disruption of one or both of the co-receptors maybe accomplished to render the cell resistant to the virus (see U.S. Pat.No. 7,951,925). Currently clinical trials are underway where HIV patientT cells are edited at the CCR5 locus ex vivo to knock out the CCR5 gene.These cells are then re-introduced into the patient to treat HIV. Thus,the methods and compositions of the invention may be used to disruptCCR5 alleles with a CRISPR/Cas system where the single guide RNAcomprises sequences to target a human CCR5 gene(chr3:46411633-46417697), especially at or near the exon region(chr3:46414394-46415452). One especially preferred region for targetingthe CCR5 gene for knock out is the region near the delta-32 mutationregion (at or near chr3:46414923-46415020). Another especially preferredregion is around the chr3: 46414522-46414643, which encodes part of thesecond extracellular loop of the CCR5 protein. The region at or near theATG protein translation initiation site (at or nearchr3:46414347-46414466) is also especially preferred for genomemodification, such as fusion of a C34 peptide to the N-terminus of CCR5by targeted integration for anti-HIV therapy. Similar studies are inprogress in animal models of CXCR4-dependent HIV where the CXCR4 isselectively disrupted, or disrupted in tandem with CCR5 to prevent HIVinfection of T cells (see U.S. Patent Publication No. 20100291048).Thus, the methods and compositions of the invention may be used todisrupt CXCR4 alleles with a CRISPR/Cas system where the single guideRNA comprises sequences to target a human CXCR4 gene(chr2:136871919-136875725), especially at or near the exon 2 region(chr2:136872439-136873482) and the region surrounding the small exon1(chr2:136875616-136875630). One preferred region for targeting the CXCR4gene for knock out is the region at or near chr2:136872863-136872982that is an analog to the delta-32 mutation region in CCR5 gene. Theregion at or near chr2:136875540-136875687 near the ATG proteintranslation initiation site of exon1 is especially preferred, and theregion at or near chr2:136873389-136873558 near the splicing site ofexon2 is especially preferred for gene modification, such as fusion of aC34 peptide to the N-terminus of CXCR4 by targeted integration foranti-HIV therapy. Thus, a sgRNA can be designed to bind to sequencesanywhere in the CCR5 or CXCR4 locus, including, but not limited to, asequence in one or more of these preferred targeting regions.

Another receptor of interest is the glucocorticoid receptor (see U.S.Patent Publication US20080188000). Knock out of this receptor inspecific therapeutic treatments allows the use of steroids that arenormally taken up by the glucocorticoid receptor. Thus, the receptor maybe targeted by a CRISPR/Cas system using the sgRNAs of the invention totarget at or near chromosome 5: 142646254-142783254. Especiallypreferred is to target the exonic sequences, for example, at or nearchromosome 5 142646255-142783254 (exon 1), 142782776-142783254 (exon 2),142779221-142780417, and especially useful 142657496-142658976 (exon 3),142693567-142693733 (exon 4), 142689662-142689778 (exon 5),142680050-142680328 (exon 6), 142678233-142678377 (exon 7),142675025-142675155 (exon 8), 142662133-142662290 (exon 9).

Specific nucleases can also be engineered to insert a peptide fusioninhibitor on to an HIV receptor to prevent HIV infection of T cells (seeco-owned US patent publication no. 20120093787), where an example ofsuch a peptide fusion inhibitor is C34 or fuzeon. Similarly HIV can betreated by using engineered nucleases to insert anti-HIV transgenes insafe harbor loci within the cell to combat the virus. Examples of suchanti-HIV genes may be selected from the group consisting of a sequenceencoding a zinc finger transcription factor that represses an HIVpolyprotein, a sequence encoding a zinc finger transcription factor thatrepresses expression of an HIV receptor, a CCR5 ribozyme, an siRNAsequence targeted to an HIV polyprotein, a sequence encoding aTrim5alpha (Trim5α) restriction factor, a sequence encoding an APOBEC3Grestriction factor, a sequence encoding a RevM10 protein, a sequenceencoding C46, other anti-HIV genes, a suicide cassette and combinationsthereof. Thus, the methods and compositions of the invention may be usedto treat or prevent HIV with a CRISPR/Cas system where the single guideRNA comprises sequences to target the CCR5 or CXCR4 gene for integrationof a suitable anti-HIV transgene. Additional non-limiting examples ofsequences suitable for targeting are shown in Table 1.

Genomic editing as described herein may be performed on any endogenoustarget. In certain embodiments, genomic modifications (e.g., transgeneintegration) are at a “safe harbor” gene. Specific “safe harbor”locations in the genome may be utilized for transgene integration thatmay either utilize the promoter found at that safe harbor locus, orallow the expressional regulation of the transgene by an exogenouspromoter that is fused to the transgene prior to insertion. Several such“safe harbor” loci have been described, including the AAVS1 (also knownas PPP1R12C,) and CCR5 genes in human cells, Rosa26 and albumin (seeco-owned U.S. Patent Publication Nos. 20080299580, 20080159996 and201000218264 and U.S. application Ser. Nos. 13/624,193 and 13/624,217)and Zp15 in plants (see U.S. Pat. No. 8,329,986). An exogenous nucleicacid or transgene sequence can comprise, for example, one or more genesor cDNA molecules, or any type of coding or noncoding sequence, as wellas one or more control elements (e.g., promoters). In addition, theexogenous nucleic acid sequence may produce one or more RNA molecules(e.g., small hairpin RNAs (shRNAs), inhibitory RNAs (RNAis), microRNAs(miRNAs), etc.). The exogenous nucleic acid sequence is introduced intothe cell such that it is integrated into the genome of the cell (e.g.the PPP1R12C gene, which lies on chromosome 19, i.e.chr19:55602840-55624858). Integration of exogenous sequences can proceedthrough both homology-dependent and homology-independent mechanisms.Thus, the methods and compositions of the invention may be used toinsert transgenes into a safe harbor locus with a CRISPR/Cas systemwhere the single guide RNA comprises sequences to target the safe harborgene. Non-limiting examples of sequences suitable for targeting areshown in Table 1.

As described above, nucleases (e.g., CRISPR/Cas) specific for the safeharbor can be utilized such that the transgene construct is inserted byeither HDR- or NHEJ-driven processes. Hypoxanthine-guaninephosphoribosyltransferase (HPRT) is an enzyme involved in purinemetabolism encoded by the HPRT1 gene (chrX:133594175-133634698). HPRT1is located on the X chromosome, and thus is present in single copy inmales. HPRT1 encodes the transferase that catalyzes the conversion ofhypoxanthine to inosine monophosphate and guanine to guanosinemonophosphate by transferring the 5-phosphorobosyl group from5-phosphoribosyl 1-pyrophosphate to the purine. The enzyme functionsprimarily to salvage purines from degraded DNA for use in renewed purinesynthesis. In the presence of 6-TG, HPRT is the enzyme responsible forthe integration of 6-TG into DNA and RNA in the cell, resulting inblockage of proper polynucleotide synthesis and metabolism. Thus, 6-TGcan be used as a selection agent to kill cells with a functional HPRTenzyme, and in addition, 6-TG can be given to cause mild immunoablationin subjects in need thereof. In a patient receiving a stem cell graft(e.g. hematopoietic or progenitor stem cells), a transgene of interestcan be integrated into the HPRT locus, knocking out the HPRT1 gene. Sucha cell population will be resistant to 6-TG toxicity. Thus when thetransgene(+)/HPRT/(−) cells are infused into the patient, a mild courseof 6-TG may increase engraftment of the cells, and those cells thatengraft will have a greater percentage of transgene integration.

HPRT has been targeted traditionally as a safe harbor for transgeneintegration (see for example Jasin et al (1996) Proc Natl Acad Sci USA93, p. 8804). It is constitutively expressed at a low level, anddisruption of the HPRT gene can be selected for both in vitro and invivo using 6-TG. However, integration into an HPRT locus via randomintegration can be difficult and occurs only at a low frequency. Use ofspecific nucleases will allow improved targeting of the HPRT locus.Thus, the methods and compositions of the invention may be used toinsert transgenes into a HPRT locus with a CRISPR/Cas system where thesingle guide RNA of the invention comprises sequences to target the safeharbor gene. Preferred is targeting the HPRT gene(chrX:133594175-133634698). Especially preferred is to target sequencesin intron 1 (at or near chromosome X: 133597660-133597662,133603544-133603546 and 133604282-133604284). Also preferred is cleavingin exon 3, especially at or near chromosome x: 133609240-133609242.Another preferred location is in the active domain of the enzyme at ornear chromosome X: 133627552-133627554. Thus, a sgRNA can be designed tobind to sequences anywhere in the HPRT locus, including, but not limitedto, a sequence in one or more of these preferred targeting regions.Additional non-limiting examples of sequences suitable for targeting areshown in Table 1.

Another useful gene to target with the methods and compositions of theinvention is the IL2 receptor common gamma chain (encoded by the IL2RGgene, at or near chrX:70327254-70331481). The IL2RG protein is thecommon receptor chain in several cytokine receptors, and mutations inthis gene are associated with X-linked severe combined immunodeficiency(“X-SCID”). Thus, targeting this gene with a CRISPR/Cas system and thesgRNAs of the invention to facilitate gene correction would bebeneficial to X-SCID patients. Preferred is to target near the end ofexon 1 (at or near Chr x: 70331274-70331276). Especially preferred is totarget intron 1 at or near chromosome x: 70331196-70331198, or in exon5, at or near chromosome x: 70329158-70329160.

The albumin gene is highly expressed in the liver. Thus, insertion of atransgene into the endogenous albumin locus using a specific nucleaseresults in high level expression of the protein or gene product encodedby that transgene, which may also be secreted into the blood stream Thetransgene can encode any protein or peptide including those providingtherapeutic benefit. For example, the transgene can encode a proteininvolved in disorders of the blood, for example, clotting disorders, anda variety of other monogenic diseases. The transgene can be insertedinto the endogenous albumin locus such that expression of the transgeneis controlled by the albumin expressional control elements, resulting inliver-specific expression of the transgene encoded protein at highconcentrations. Proteins that may be expressed may include clottingfactors such as Factor VII, Factor VIII, Factor IX, Factor X, Factor XI,Factor XIII, vWF and the like, antibodies, proteins relevant tolyososomal storage, insulin, alpha 1-antitrypsin, and indeed any peptideor protein that when so expressed provides benefit. Thus, the methodsand compositions of the invention may be used to insert transgenes intoan albumin gene with a CRISPR/Cas system where the single guide RNAcomprises sequences to target the albumin gene (at or nearchr4:74269972-74287129). Preferred targets are those in the exonicsequences, for example at or near chr:4 74270073-74270883 (exon 1,especially preferred, most preferred at chromosome 4:74270385-74270485), 74270840-74272396 (exon 2, especially preferred ator near chromosome 4: 74270840-74272396), 74272428-74274361 (exon 3),74274472-74275122 (exon 4), 74275154-74276079 (exon 5),74276076-74277763 (exon 6), 74277792-74279187 (exon 7),74279301-74280802 (exon 8), 74280834-74282023 (exon 9),74282020-74283298 (exon 10), 74283336-74283855 (exon 11),74283978-74285274 (exon 12, especially preferred), 74285306-74286021(exon 13, especially preferred), and 74285988-74286859 (exon 14,especially preferred). Thus, a sgRNA can be designed to bind tosequences anywhere in the albumin locus, including, but not limited to,a sequence in one or more of these preferred targeting regions.Additional non-limiting examples of sequences suitable for targeting areshown in Table 1.

Lysosomal storage diseases (LSDs) are a group of rare metabolicmonogenic diseases characterized by the lack of functional individuallysosomal proteins normally involved in the breakdown of waste lipids,glycoproteins and mucopolysaccharides. These diseases are characterizedby a buildup of these compounds in the cell since it is unable toprocess them for recycling due to the mis-functioning of a specificenzyme. The most common examples are Gaucher's (glucocerebrosidasedeficiency—gene name: GBA), Fabry's (α galactosidase deficiency—GLA),Hunter's (iduronate-2-sulfatase deficiency—IDS), Hurler's (alpha-Liduronidase deficiency—IDUA), and Niemann-Pick's (sphingomyelinphosphodiesterase 1deficiency—SMPD1) diseases. When grouped alltogether, LSDs have an incidence in the population of about 1 in 7000births. These diseases have devastating effects on those afflicted withthem. They are usually first diagnosed in babies who may havecharacteristic facial and body growth patterns and may have moderate tosevere mental retardation. Treatment options include enzyme replacementtherapy (ERT) where the missing enzyme is given to the patient, usuallythrough intravenous injection in large doses. Such treatment is only totreat the symptoms and is not curative, thus the patient must be givenrepeated dosing of these proteins for the rest of their lives, andpotentially may develop neutralizing antibodies to the injected protein.Often these proteins have a short serum half-life, and so the patientmust also endure frequent infusions of the protein. For example,Gaucher's disease patients receiving the Cerezyme® product(imiglucerase) must have infusions three times per week. Production andpurification of the enzymes is also problematic, and so the treatmentsare very costly (>$100,000 per year per patient). Thus, the specificnucleases of the invention may be used to insert genes encoding themissing wild type versions of the proteins involved in lysosomal storagediseases into safe harbor loci such as albumin for expression andtreatment of the LSD.

Hemophilia B is a genetic disorder of the blood-clotting system,characterized by bleeding into joints and soft tissues, and by excessivebleeding into any site experiencing trauma or undergoing surgery. Whilehemophilia B is clinically indistinguishable from hemophilia A, factorVIII (FVIII) is deficient or absent in hemophilia A and factor IX (FIXor F.IX) is deficient or absent in patients with hemophilia B. Factor IXencodes one of the serine proteases involved with the coagulationsystem, and it has been shown that restoration of even 3% of normalcirculating levels of wild type Factor IX protein can preventspontaneous bleeding.

Gene therapy, including liver-directed gene therapy protocols and directintramuscular injection, involving the introduction of plasmid and othervectors (e.g., AAV) encoding a functional FIX protein have beendescribed for treatment of hemophilia B. See, e.g., U.S. Pat. No.6,936,243; Lee et al. (2004) Pharm. Res. 7:1229-1232; Graham et al.(2008) Genet Vaccines Ther. 3:6-9. However, in these protocols, theformation of inhibitory anti-factor IX (anti-FIX) antibodies andantibodies against the delivery vehicle remains a major complication ofFIX protein replacement-based treatment for hemophilia B. Thus, use ofdesigned nucleases and related technology to either correct theendogenous Factor VIII or Factor IXgene or introduce a wild type copy ofthe gene in a safe harbor locus can restore clotting activity in atreated patient. A preferred targeting region for the Factor VIII geneis at or near the gene sequence located at chrX:154064064-154250998.Especially preferred for targeting the Factor VIII gene is the region ofintron 22, which is a site of common inversion evens that account for upto 50% of FVIII mutations in severe hemophilia A patients, located at ornear chrX:154091503-154124351. For the Factor IX (F.IX) gene, preferredfor targeting is the gene sequence located at or nearchrX:138,612,895-138,645,617. Especially preferred are targets inregions in intron 1, e.g. at or near chrX:138613760-138613816,chrX:138618832-138618887 and/or chrX:138613815-138613872. Thus, a sgRNAcan be designed to bind to sequences anywhere in the Factor VIII orFactor IX loci, including, but not limited to, a sequence in one or moreof these preferred targeting regions.

Parkinson's disease (PD) is a neurodegenerative disease that afflictsapproximately 4-6 million people worldwide. In the United States,approximately one to two hundred people per 100,000 have PD. Theprevalence of PD increases in the older population, with approximately4% of people over the age of 80 suffering from this disease (Davie(2008) Brit Med Bull 86(1) p. 109), although 10% of patients are under40 years of age (Kumari (2009) FEBS J. 276(22) p. 6455).

It appears that many factors can play a role in disease onset and/orprogression of PD. For example, genetic mutations in the leucine richrepeat kinase 2 gene (LRRK2, also known as PARKS) has been identified tobe involved in both familial and sporadic forms of PD. In fact, studiessuggest that LRRK2 mutations may be responsible for between 5 and 13% offamilial PD, and from 1 to 5% of sporadic PD. The protein itself is alarge (>280 kD) multidomain protein containing the following knowndomains: armadillo (ARM), ankryn (ANK), LRR, Ras of complex proteins(ROC), C-terminal of ROC (COR), mitogen-activated protein kinase kinasekinase and WD40. Thus, LRRK2 contains several protein-proteininteractive domains (ARM/ANK, LRR and WD40) suggesting that LRRK2 playsa role in protein complex formation (Kumari, ibid). Several clusters ofmutations have been identified which fall across its length of the gene,with the majority of pathological mutations clustering in the enzymaticdomains of the protein.

Specifically, the LRRK2 mutation G2019S has been suggested to play animportant role in PD in some ethnicities. The mutation is autosomaldominant and the lifetime penetrance for the mutation has been estimatedat 31.8%. The SNP responsible for this missense mutation in patients isannotated as rs34637584 in the human genome, and is a G to Asubstitution at the genomic level (6055G>A). This LRRK2 mutation can bereferred to either as G2019S or 6055G>A and is found at or nearchr12:40734202. The G2019S mutation has been shown to increase LRRK2kinase activity, and is found in the within the activation domain orprotein kinase-like domain of the protein (Luzon-Toro (2007) Hum MolGenet 16(17) p. 2031). Thus, a specific nuclease can be used to correcta mutation in the LRRK2 gene for the treatment of PD. Thus, the methodsand compositions of the invention may be used to correct a LRRK2 genewith a CRISPR/Cas system where the single guide RNA comprises sequencesto target the LRRK2 gene Especially preferred is a sgRNA designed totarget at or near chr12:40734202-40734202. Non-limiting examples ofsequences suitable for targeting are shown in Table 1.

Trinucleotide repeat expansion disorders were first characterized in theearly 1990s (see Di Prospero and Fischbeck, (2005) Nature ReviewsGenetics vol 6: 756-765). These disorders involve the localizedexpansion of unstable repeats of sets of three nucleotides and canresult in loss of function of the gene in which the repeat resides, again of toxic function, or both. Trinucleotide repeats can be located inany part of the gene, including non-coding and coding gene regions.Repeats located within the coding regions typically involve either arepeated glutamine encoding triplet (CAG) or an alanine encoding triplet(CGA). Expanded repeat regions within non-coding sequences can lead toaberrant expression of the gene while expanded repeats within codingregions (also known as codon reiteration disorders) may causemis-folding and protein aggregation.

Huntington's Disease (HD), also known as Huntington's Chorea, is aprogressive disorder of motor, cognitive and psychiatric disturbancesThe mean age of onset for this disease is age 35-44 years, although inabout 10% of cases, onset occurs prior to age 21, and the averagelifespan post-diagnosis of the disease is 15-18 years. Prevalence isabout 3 to 7 among 100,000 people of western European descent. Thedisease is associated with expansion of the CAG repeat region in theendogenous Huntintin gene (Htt, chr4:3076237-3245687). Normal Httalleles contain 15-20 CAG repeats, while alleles containing 35 or morerepeats can be considered potentially HD causing alleles and confer riskfor developing the disease. Alleles containing 36-39 repeats areconsidered incompletely penetrant, and those individuals harboring thosealleles may or may not develop the disease (or may develop symptomslater in life) while alleles containing 40 repeats or more areconsidered completely penetrant and no asymptomatic persons containingHD alleles with this many repeats have been reported. Those individualswith juvenile onset HD (<21 years of age) are often found to have 60 ormore CAG repeats. Thus, the methods and compositions of the inventionmay be used to disrupt a Htt allele with a CRISPR/Cas system where thesingle guide RNA comprises sequences to target the expanded Htt gene.Especially preferred is targeting chromosome 4, at or near3071604-3081660. The methods and compositions of the invention may alsobe used to selectively repress a mutant Htt allele using a CRISPR/Cassystem fusion protein comprising a repression domains. Non-limitingexamples of sequences suitable for targeting are shown in Table 1.

Retinitis pigmentosa (RP) refers to a diverse group of hereditarydiseases affecting two million people worldwide that lead to incurableblindness. RP is one of the most common forms of inherited retinaldegeneration, and there are multiple genes whose mutation can lead toRP. More than 100 mutations in 44 genes expressed in rod photoreceptorshave thus far been identified, accounting for 15% of all types ofretinal degeneration, most of which are missense mutations and areusually autosomal dominant.

Rhodopsin is a pigment of the retina that is involved in the firstevents in the perception of light. It is made of the protein moietyopsin covalently linked to a retinal cofactor. Rhodopsin is encoded bythe RHO gene (chr3:129247482-129254187), and the protein has a molecularweight of approximately 40 kD and spans the membrane of the rod cell.The retinal cofactor absorbs light as it enters the retina and becomesphotoexcited, causing it to undergo a change in molecular configuration,and dissociates from the opsin. This change initiates the process thateventually causes electrical impulses to be sent to the brain along theoptic nerve. In relation to RP, more than 80 mutations in the rhodopsingene have been identified that account for 30% of all Autosomal DominantRetinitis Pigmentosa (ADRP) in humans (Dryj a et al (2000) InvestOpthalmol Vis Sci 41: 3124-3127). Three point mutations in the humanrhodopsin gene (leading to P23H, Q64X and Q344X in the protein sequence)are known to cause ADRP in humans. See, e.g., Olsson et al. (1992)Neuron 9(5):815-30. The P23H mutation is the most common rhodopsinmutation in the United States. Due to problems with protein folding,P23H rhodopsin only partially reconstitutes with retinal in vitro (Liuet al (1996) Proc Nat'l Acad Sci 93:4554-4559), and mutant rhodopsinexpressed in transgenics causes retinal degeneration (Goto et al (1995)Invest Opthalmol Vis Sci 36:62-71). Thus, the methods and compositionsof the invention may be used to disrupt a RHO allele with a CRISPR/Cassystem where the single guide RNA comprises sequences to target a humanRHO gene (at or near chr3:129247482-129254187). Targeting RHO atspecific locations is useful for facilitating gene correction. Preferredtarget locations include exon 1 (at or near chr3:129247577-129247937 forcorrecting the sequence associated with the P23H or Q64X mutations), andexon5 (at or near chr3:129251376-129251615). Non-limiting examples ofsequences suitable for targeting are shown in Table 1.

Lung diseases, including inherited disorders such as Cystic Fibrosis(CF) and Surfactant Protein B (SP-B) Deficiency remain an issue inpediatric populations. SP-B deficiency is a rare lung disease whereprotein and fat molecules accumulate in the distant parts of the lungsand affect breathing. The disease is caused by a deficiency of the lungsurfactant protein B, primarily due to a defect in the SFTPB gene(located at or near chr2:85884440-85895374) which encodes thepulmonary-associated surfactant B protein (SPB), an amphipathicsurfactant protein essential for lung function and homeostasis afterbirth. The most common mutation in SP-B deficiency is a mutationdesignated “121ins2” which results in the nucleotide “C” at position 131in the mRNA being converted into “GAA. (in the genomic sequence, thiscorresponds to 375C-GAA and is located in exon 4). Thus, the methods andcompositions of the invention can be used to targets a CRISPR/Cas systemusing a sgRNA of the invention to the SFTPB gene at or nearchr2:85884440-85895374, and most preferably, to exon 4 (at or nearchr2:85893740-85893865).

CF is an autosomal recessive disorder affecting 1 in 1500 to 4000 livebirths, and is one of the most common inherited pediatric disorders. Theprimary defect in CF is in the regulation of epithelial chloridetransport by a chloride channel protein encoded by the cystic fibrosistransmembrane conductance regulator (CFTR) gene (locatedchr7:117120017-117308718). See, e.g., Kerem et al. (1989) Science245:1073-1080; Kreda et al. (2005) Mol Biol Cell 16:2154-2167. About 70%of mutations observed in CF patients result from deletion of three basepairs in CFTR's nucleotide sequence, resulting in the loss of the aminoacid phenylalanine located at position 508 in the protein (a mutationreferred to as ΔF508, (located in exon 11). In a wild type genome, aminoacid 507 is an isoleucine, and is encoded by the codon TAG where the Gis nucleotide 1652 in the gene. Amino acid 508 is a phenylalanine,encoded by AAA. In the Δ508 mutation, the G from the 507 codon isdeleted along with the first two As of the 508 codon, such that themutation has the sequence TAA at the deleted 507-508 encoding position.TAA also encodes an isoleucine, but the phenylalanine at wild typeposition 508 is lost. For the ΔI507 deletion, either the isoleucine atposition 506 or 507 is deleted. For this mutation, the nucleotides at1648-1650 or 1651-1653 are lost, or some combination thereof to resultin only one isoleucine in the resultant protein. Compound (heterozygous)mutations (ΔF508 and ΔI507) have also been documented. See, e.g., Orozcoet al. (1994) Am J Med Genet. 51(2):137-9. CF patients, either compoundheterozygous ΔI507/ΔF508 or homozygous ΔF508/ΔF508, fail to express thefully glycosylated CFTR protein and the partially glycosylated proteinis not expressed on the cell surface (see, e.g., Kreda et al. (2005) MolBiol Cell 16:2154-2167; Cheng et al. (1990) Cell 63:827-834) as isrequired for CFTR function. Individuals bearing either the ΔI507 orΔF508 CFTR mutations at only one allele (i.e. wt/ΔI507 or wt/ΔF508) areCF carriers and exhibit no defects in lung cell function. See, e.g.,Kerem et al. (1990) Proc Natl Acad Sci USA 87:8447-8451. Thus, themethods and compositions of the invention may be used to disrupt orcorrect a CFTR allele with a CRISPR/Cas system where the single guideRNA comprises sequences to target a human CFTR gene. Especiallypreferred for targeting is at or near chr7:117227793-117227887).Non-limiting examples of sequences suitable for targeting are shown inTable 1.

Muscular dystrophies are diseases that are characterized by aprogressive degeneration and weakening of muscle groups. One well knownmuscular dystrophy is Duchenne's muscular dystrophy, which is anX-linked disease that afflicts 1 in every 3500 boys. It is caused by thelack of the protein dystrophin in the individual muscle cells, andsymptoms first appear when the child is approximately 3 years old, anddepending on the severity of the disease, death can occur when thepatient is in his twenties. The gene encoding dystrophin, DMD (locatedat or near chrX:31137345-33229673), is extremely large and covers 2.4megabases of DNA comprising 79 exons that encode a 14 kb mRNA. Inpatients that lack functional dystrophin, approximately 40% have pointmutations that cause a frameshift in the coding sequence such thatduring translation, a premature stop is encountered resulting in theproduction of a truncated or non-functioning protein. The other 60% havelarge insertions or deletions that also result in alteration of frameand similarly result in production of a non-functional protein (Nowakand Davies (2004) EMBO Reports 5(9): 872-876). Patients withnon-functional dystrophin have the most severe disease, which is alsoknown as Duchenne's Muscular Dystrophy (DMD). Other patients, whosemutations are characterized by gene deletions of regions that encodeinternal portions of dystrophin, resulting in less functional dystrophinprotein as compared to wild type, may have less severe disease, which iscalled Becker Muscular Dystrophy (BMD). BMD patients have been known tolive into their 50's. Targeting the DMD gene may be useful for genecorrection of point mutations or for changing splicing patterns of themRNA transcript, allowing the cell to produce a functional dystrophinprotein. Thus, a sgRNA can be designed to bind to sequences anywhere inthe DMD locus, at or near chrX:31137345-33229673. Adoptive immunotherapyis the practice of achieving highly specific T cell stimulation of acertain subpopulation of CTLs that possess a high-avidity TCR to thetumor antigen, stimulating and expanding them ex vivo, and thenintroducing them into the patient. Adoptive immunotherapy isparticularly effective if native lymphocytes are removed from thepatient before the infusion of tumor-specific cells. The idea behindthis type of therapy is that if the introduced high-avidity CTLs aresuccessful, once the tumor has been cleared, some of these cells willremain as memory T cells and will persist in the patient in case thecancer reappears. However, transfer of any TCR transgenes into host Tcells carries with it the caveats associated with most gene transfermethods, namely, unregulated and unpredictable insertion of the TCRtransgene expression cassette into the genome, often at a low level.Such poorly controlled insertion of the desired transgene can result ineffects of the transgene on surrounding genes as well as silencing ofthe transgene due to effects from the neighboring genes. In addition,the endogenous TCR genes that are co-expressed in the T cell engineeredwith the introduced TCR transgene could cause undesired stimulation ofthe T cell by the antigen recognized by the endogenous TCR, undesiredstimulation of the T cell by unintended antigens due to the mispairingof the TCR transgene with the endogenous TCR subunits creating a novelTCR complex with novel recognition properties, or can lead to suboptimalstimulation against the antigen of interest by the creation of inactiveTCRs due to heterodimerization of the transgene encoded TCR subunitswith the endogenous TCR proteins. In fact, the risk of severe autoimmunetoxicity resulting from the formation of self-reactive TCR frommispairing of endogenous and exogenous chains has been recentlyhighlighted in a murine model (Bendle et al. (2010) Nature Medicine16:565-570) and in human cells (van Loenen et al. (2010) Proc Nall AcadSci USA 107:10972-7). Additionally, the tumor-specific TCR may beexpressed at suboptimal levels on the cell surface, due to competitionwith the endogenous and mispaired TCR for the CD3 molecules, required toexpress the complex on the cell surface. Low TCR expression affects theavidity and efficacy of the transgenic T cell. Examples of targets thatcan be used to make high affinity TCRs against for introduction into a Tcell are WT1 and NYEso. Thus, the methods and compositions of theinvention may be used to disrupt or correct an endogenous TCR gene witha CRISPR/Cas system where the single guide RNA comprises sequences totarget a human TCR alpha (TRAC or TCRA, located at or nearchr6:42883727-42893575) or beta (TRBC or TCRB, located at or nearchr7:142197572-142198055) gene. Non-limiting examples of sequencessuitable for targeting are shown in Table 1.

The programmed death receptor (PD1 or PD-1, also known as PDCD1) hasbeen shown to be involved in regulating the balance between T cellactivation and T cell tolerance in response to chronic antigens. Upon Tcell activation, PD1 expression is induced in T cells. The ligands forthe PD1 receptor are PD1 ligand (PDL1 also known as B7-H1 and CD272) andPDL2 (also known as B7-DC and CD273), and are normally expressed inantigen presenting cells and in the periphery. PD1-PDL (PD1 ligand)coupling causes deactivation of the T cell and is involved in inducing Tcell tolerance (see Pardoll (2012) Nat Rev 12:252). During HIV1infection, expression of PD1 has been found to be increased in CD4+ Tcells, and PDL1 expression is increased on antigen presenting cells(APCs), tipping the balance between T cell inhibition and T cellstimulation towards T cell inhibition (see Freeman et al (2006) J ExpMed 203(10):2223-2227). It is thought that PD1 up-regulation is somehowtied to T cell exhaustion (defined as a progressive loss of key effectorfunctions) when T cell dysfunction is observed in the presence ofchronic antigen exposure as is the case in HIV infection. PD1up-regulation may also be associated with increased apoptosis in thesesame sets of cells during chronic viral infection (see Petrovas et al,(2009) J Immunol. 183(2):1120-32). PD1 may also play a role intumor-specific escape from immune surveillance. It has been demonstratedthat PD1 is highly expressed in tumor-specific cytotoxic T lymphocytes(CTLs) in both chronic myelogenous leukemia (CML) and acute myelogenousleukemia (AML). PD1 is also up-regulated in melanoma infiltrating Tlymphocytes (TILs) (see Dotti (2009) Blood 114 (8): 1457-58). Tumorshave been found to express the PD1 ligand PD-L1 or, more rarely, the PD1ligand PDL2 which, when combined with the up-regulation of PD1 in CTLs,may be a contributory factor in the loss in T cell functionality and theinability of CTLs to mediate an effective anti-tumor response.Researchers have shown that in mice chronically infected withlymphocytic choriomeningitis virus (LCMV), administration of anti-PD1antibodies blocked PD1-PDL interaction and was able to restore some Tcell functionality (proliferation and cytokine secretion), leading to adecrease in viral load (Barber et al (2006) Nature 439(9): 682-687).Additionally, a fully human PD-1 specific IgG4 monoclonal antibody hasbeen tested in the clinic in an oncology setting on patients with avariety of disease backgrounds (advanced melanoma, renal cell carcinoma,non-small cell lung cancer, colorectal cancer or prostate cancer).Clinical activity was observed in melanoma, renal cell and non-smallcell lung cancer patients and preliminary data suggested that detectionof PD 1 ligand expression by the tumor prior to treatment correlatedwith clinical outcome (see Wolfe (2012) Oncology Business Review, July;and Pardoll, ibid). Thus, the methods and compositions of the inventionmay be used to disrupt a PD1 allele with a CRISPR/Cas system where thesingle guide RNA comprises sequences to target a human PD1 gene(chr2:242792033-242801058). One preferred region for targeting the PD1gene for knock out is at or near the region near that ATG proteintranslation initiation site. This corresponds to nucleotides chr2:242800981-242800982 of the PD1 gene. Especially preferred are targetsites the PD1 gene where the PAM position for the S. pyogenes Cas9 islocated at or near chromosome 2 at position 242800980-242800981,242800975-242800976, 242800971-242800972, 242800970-242800971,242800967-242800968, and 242800965-242800966. Another especiallypreferred targeting location is around the region near the PD-1 ligandbinding domain (chromosome 2, nucleotides 242794834-242794835 and242794828-242794829). Another preferred region for targeting is at ornear the region near the immunoreceptor tyrosine-based switch motif(e.g., chromosome 2 242793349-242793350, 242793338-242793339,242793330-242793331 or 242793327-242793328). Mutations in this regiondisable PD1 function. Especially preferred are target sites for the S.pyogenes Cas9 protein at positions at or near chromosome 2:242800953-242800979, 242794976-242795005, 242794416-242794444 and242793405-242793433. A preferred targeting region for the Factor VIIIgene is at or near the gene sequence located atchrX:154064064-154250998. Especially preferred for targeting the FactorVIII gene is the region of intron 22, which is a site of commoninversion evens that account for up to 50% of FVIII mutations in severehemophilia A patients, located at or near chrX:154091503-154124351. Forthe Factor IX (F.IX) gene, preferred for targeting is the gene sequencelocated at or near chrX:138,612,895-138,645,617. Especially preferredare targets in regions in intron 1, e.g. at or nearchrX:138613760-138613816, chrX:138618832-138618887 and/orchrX:138613815-138613872. Thus, a sgRNA can be designed to bind tosequences anywhere in the PD1 locus, including, but not limited to, asequence in one or more of these preferred targeting regions.Non-limiting examples of sequences suitable for targeting are shown inTable 1.

Another modulator of T cell activity is the CTLA-4 receptor. Similar tothe T cell receptor CD28, CTLA-4 interacts with the CD80 and CD86ligands on antigen presenting cells. But while interaction of theseantigens with CD28 causes activation of T cells, interaction of CD80 orCD86 with CTLA-4 antagonizes T-cell activation by interfering with IL-2secretion and IL-2 receptor expression, and by inhibiting the expressionof critical cell cycle components. CTLA-4 is not found on the surface ofmost resting T cells, but is upregulated transiently after T-cellactivation. Thus CTLA-4 is also involved in the balance of activatingand inhibiting T cell activity (see Attia et al (2005) J Clin Oncol.23(25): 6043-6053). Initial clinical studies involving the use of CTLA 4antibodies in subjects with metastatic melanoma found regression of thedisease (Attia, ibid), but later studies found that subject treated withthe antibodies exhibited side effects of the therapy (immune-relatedadverse events: rashes, colitis, hepatitis etc.) that seemed to berelated to a breaking of self-tolerance. Analysis of this data suggestedthat greater tumor regression as a result of the anti-CTLA4 antibodycorrelated directly with a greater severity of immune-related adverseevents (Weber (2007) Oncologist 12(7): 864-872). Thus, the methods andcompositions of the invention may be used to disrupt a CTLA-4 gene witha CRISPR/Cas system where the single guide RNA comprises sequences totarget a human CTLA-4 gene, located at or near chr2:204732511-204738683.Non-limiting examples of sequences suitable for targeting are shown inTable 1.

Chimeric Antigen Receptors (CARs) are molecules designed to targetimmune cells to specific molecular targets expressed on cell surfaces.In their most basic form, they are receptors introduced to a cell thatcouple a specificity domain expressed on the outside of the cell tosignaling pathways on the inside of the cell such that when thespecificity domain interacts with its target, the cell becomesactivated. Often CARs are made from variants of T-cell receptors (TCRs)where a specificity domain such as a scFv or some type of receptor isfused to the signaling domain of a TCR. These constructs are thenintroduced into a T cell allowing the T cell to become activated in thepresence of a cell expressing the target antigen, resulting in theattack on the targeted cell by the activated T cell in a non-MHCdependent manner (see Chicaybam et al (2011) Int Rev Immunol30:294-311). Currently, tumor specific CARs targeting a variety of tumorantigens are being tested in the clinic for treatment of a variety ofdifferent cancers. Examples of these cancers and their antigens that arebeing targeted includes follicular lymphoma (CD20 or GD2), neuroblastoma(CD171), non-Hodgkin lymphoma (CD20), lymphoma (CD19), glioblastoma(IL13Rα2), chronic lymphocytic leukemia or CLL and acute lymphocyticleukemia or ALL (both CD19). Virus specific CARs have also beendeveloped to attack cells harboring virus such as HIV. For example, aclinical trial was initiated using a CAR specific for Gp100 fortreatment of HIV (Chicaybam, ibid).

As useful as it is to develop a technology that will cause a T cell tore-direct its attention to specific cells such as cancer cells, thereremains the issue that these target cells often express of PD-1 ligand.As such, the PD1-PD-L1/PD-L2 interaction enables the tumor to escapeaction by the CAR-targeted T cell by deactivating the T cells andincreasing apoptosis and cell exhaustion. Additionally, the PD1-PDLinteractions are also involved in the repression of the T cell responseto HIV, where increased expression of both PD1 and PDL leads to T cellexhaustion. Induction of CTLA-4 expression on activated T cells is alsoone of the first steps to damping the immune response, and thus a T cellarmed with a CAR might become inactive due to the engagement of thissystem designed to balance T cell activation with T cell inhibition.Thus, the CRISPR/Cas system of the invention can be used with the sgRNAsof the invention described above to knockout CTLA-4 and/or PD1 in a Tcell comprising a CAR.

MHC antigens were first characterized as proteins that played a majorrole in transplantation reactions. Rejection is mediated by T cellsreacting to the histocompatibility antigens on the surface of implantedtissues, and the largest group of these antigens is the majorhistocompatibility antigens (MHC). MHC proteins are of two classes, Iand II. The class I MHC proteins are heterodimers of two proteins, the αchain, which is a transmembrane protein encoded by the MHC 1 gene, andthe β2 microblogulin chain, which is a small extracellular protein thatis encoded by a gene that does not lie within the MHC gene cluster. Theα chain folds into three globular domains and when the β2 microglobulinchain is associated, the globular structure complex is similar to anantibody complex. The foreign peptides are presented on the two mostN-terminal domains which are also the most variable. Class II MHCproteins are also heterodimers, but the heterodimers comprise twotransmembrane proteins encoded by genes within the MHC complex. Theclass I MHC:antigen complex interacts with cytotoxic T cells while theclass II MHC presents antigens to helper T cells. In addition, class IMHC proteins tend to be expressed in nearly all nucleated cells andplatelets (and red blood cells in mice) while class II MHC protein aremore selectively expressed. Typically, class II MHC proteins areexpressed on B cells, some macrophage and monocytes, Langerhans cells,and dendritic cells.

The class I HLA gene cluster in humans comprises three major loci, B, Cand A, as well as several minor loci. HLA-A, H:A-B and HLA-C are the HLAclass I heavy chain paralogues. The class I molecule is a heterodimerconsisting of a MHC alpha heavy chain (HLA-A, HLA-B or HLA-C) and alight chain (beta-2 microglobulin). The heavy chain is anchored in themembrane. Class I molecules play a central role in the immune system bypresenting peptides derived from the endoplasmic reticulum lumen. Theheavy chain is approximately 45 kDa and its gene contains 8 exons. Exon1 encodes the leader peptide, exons 2 and 3 encode the alpha1 and alpha2domains, which both bind the peptide, exon 4 encodes the alpha3 domain,exon 5 encodes the transmembrane region, and exons 6 and 7 encode thecytoplasmic tail. Polymorphisms within exon 2 and exon 3 are responsiblefor the peptide binding specificity of each class one molecule. Apreferred region for targeting an sgRNA in HLA-A, B or C is within thegene sequence, (e.g. chr6:29910247-29912868 for HLA-A,chr6:31236526-31239913 for HLA-B, and chr6:31236526-31239125 for HLA-C).Especially preferred is targeting sequences within the leader sequence(e.g., at or near chr6:31324936-31324989 for HLA-B), the alpha 1 andalpha 2 domains (e.g. at or near chr6:31324863-31324935 andchr6:31324735-31324862, respectively for HLA-B) and the alpha3 domain(e.g. at or near chr6:31324465-31324734 for HLA-B).

The class II HLA cluster also comprises three major loci, DP, DQ and DR,and both the class I and class II gene clusters are polymorphic, in thatthere are several different alleles of both the class I and II geneswithin the population. HLA-DPA1, HLA-DQA1 and HLA-DRA belong to the HLAclass II alpha chain paralogues. This class II molecule is a heterodimerconsisting of an alpha (DPA) and a beta (DPB) chain, both anchored inthe membrane. They play a central role in the immune system bypresenting peptides derived from extracellular proteins. Class IImolecules are expressed in antigen presenting cells (APC: B lymphocytes,dendritic cells, macrophages). The alpha chains are approximately 33-35kDa and their genes (chr6_ssto_hap7:3,754,283-3,759,493 for HLA-DRA,chr6:32605183-32611429 for HLA-DQ and chr6:33032346-33048555 forHLA-DPA) contain 5 exons. Exon one (e.g. at or nearchr6:33041248-33041347 for HLA-DPA1) encodes the leader peptide, exons 2and 3 (e.g. at or near chr6:33037418-33037663 and chr6:33036796-33037077for HLA-DPA1) encode the two extracellular domains, exon 4 (e.g. at ornear hr6:33036427-33036581 for HLA-DPA1) encodes the transmembranedomain and the cytoplasmic tail. Thus, especially preferred regions totarget with a CRIPSR-Cas system of the invention are within exons one,two and three. Within the DP molecule both the alpha chain and the betachain contain the polymorphisms specifying the peptide bindingspecificities, resulting in up to 4 different molecules. Thus, a sgRNAcan be designed to bind to sequences anywhere in the HLA-DPA1, HLA-DQ1or HLA-DRA loci, including, but not limited to, a sequence in one ormore of these preferred targeting regions.

There are also several accessory proteins that play a role in HLAfunctioning as well. The Tap1 (encoded at chr6:32812986-32821748) andTap2 (encoded at chr6:32793187-32806547) subunits are parts of the TAPtransporter complex that is essential in loading peptide antigens on tothe class I HLA complexes, and the LMP2 and LMP7 proteosome subunitsplay roles in the proteolytic degradation of antigens into peptides fordisplay on the HLA. Reduction in LMP7 (chr6_dbb_hap3:4089872-4093057)has been shown to reduce the amount of MHC class I at the cell surface,perhaps through a lack of stabilization (see Fehling et al (1999)Science 265:1234-1237). In addition to TAP and LMP, there is the tapasingene (chr6:33271410-33282164), whose product forms a bridge between theTAP complex and the HLA class I chains and enhances peptide loading.Reduction in tapasin results in cells with impaired MHC class Iassembly, reduced cell surface expression of the MHC class I andimpaired immune responses (see Grandea et al (2000) Immunity 13:213-222and Garbi et al (2000) Nat Immunol 1:234-238). Regulation of class IIMHC expression is dependent upon the activity of the MHCII enhanceosomecomplex. The enhanceosome components (one of the most highly studiedcomponents of the enhanceosome complex is the RFX5 gene product (seeVillard et al (2000) MCB 20(10): 3364-3376)) are nearly universallyexpressed and expression of these components does not seem to controlthe tissue specific expression of MHC class II genes or their IFN-γinduced up-regulation. Instead, it appears that a protein known as CIITA(class II transactivator) which is a non-DNA binding protein, serves asa master control factor for MCHII expression. In contrast to the otherenhanceosome members, CIITA does exhibit tissue specific expression, isup-regulated by IFN-γ, and has been shown to be inhibited by severalbacteria and viruses which can cause a down regulation of MHC class IIexpression (thought to be part of a bacterial attempt to evade immunesurveillance (see Leibund Gut-Landmann et al (2004) Eur. J. Immunol34:1513-1525)). Thus, the methods and compositions of the invention maybe used to disrupt an HLA gene or an HLA regulatory gene with aCRISPR/Cas system where the single guide RNA comprises sequences totarget a human HLA gene or HLA regulatory gene. A sgRNA can be designedto bind to sequences anywhere in the HLA locus, including, but notlimited to, a gene sequence encoding HLA-A, HLA-B, HLA-C, HLA-DPA,HLA-DQ or HLA-DRA, and to preferred targeting regions within these genesdiscussed above. Additionally, sgRNAs can be designed to bind tosequences in genes whose products interact with the MHC proteins,including TAP1, TAP2, LMP2, LMP7, and tapasin, or to sequences in geneswhose products regulate the expression of these genes, including thosein the MHCII enhanceosome complex (RFX5 (chr1:151313116-151319769) andCIITA (chr16:10971055-11002744). Non-limiting examples of sequencessuitable for targeting are shown in Table 1.

Disclosed herein are compositions and methods useful for modulation ofexpression and targeted cleavage and alteration of genes in plants,particularly paralogous genes in plants. Regulation of a paralogous genecan be modulated, e.g., by using engineered transcription factors ormodifying gene regulatory regions. Genes can be altered, e.g., bytargeted cleavage followed by intrachromosomal homologous recombinationor by targeted cleavage followed by homologous recombination between anexogenous polynucleotide (comprising one or more regions of homologywith the gene nucleotide sequence) and a genomic sequence. Anon-limiting example of a paralogous gene in plants is the EPSPS gene.Thus, the methods and compositions of the invention may be used todisrupt an EPSPS gene with a CRISPR/Cas system where the single guideRNA comprises sequences to target a plant EPSPS gene.

The present disclosure provides methods and compositions for expressingone or more products of an exogenous nucleic acid sequence (i.e. aprotein or a RNA molecule) that has been integrated into a Zp15 gene ina plant cell. As shown in the co-owned U.S. Pat. No. 8,329,986, theintegration of one or more exogenous sequences at or near the Zp15 locusdoes not appear to impair the ability of the host plant to regenerate,flower or produce seed and, optionally, allows heritable transmission ofthe exogenous sequence(s) over generations. The exogenous nucleic acidsequences can comprise, for example, one or more genes or cDNAmolecules, or any type of coding or noncoding sequence, as well as oneor more control elements (e.g., promoters). For instance, herbicidetolerance genes can be integrated into this locus to produce crop plantswith the desired herbicide resistance. Cells containing exogenousnucleic acids at or near the Zp15 locus can also contribute to thegametophyte (germline) and therefore be transmitted to progeny insubsequent generations. Thus, the methods and compositions of theinvention may be used to disrupt a Zp15 gene with a CRISPR/Cas systemwhere the single guide RNA comprises sequences to target a plant ZP15gene.

The present disclosure provides compositions and methods for modulatingexpression and for targeted alteration in whole plants or plant cells ofone or more plant genes involved in fatty acid biosynthesis, therebyaltering the fatty acid composition in the whole plant or plant cells.Whole plants or plant cells can be from monocotyledonous (monocots) ordicotyledonous (dicots) plant species, including in some particularembodiments oil-producing plants, and also include cultured cells, cellsin a plant at any stage of development, and plant cells that have beenremoved from a whole plant and which cells (or their descendants) willbe regenerated into plants. Plant cells can contain one or morehomologous or paralogous gene sequences, any number of which or all ofwhich can be targeted for modification by the methods disclosed herein.

In one aspect, described herein is a DNA-binding domain (e.g., Casprotein) that specifically binds to a gene involved in a plant fattyacid biosynthesis pathway. In some embodiments, the gene is a Brassicanapus gene. In some particular embodiments, the Brassica napus gene canencode Acetyl-COA carboxylase (ACCase), β-ketoacyl-ACP synthetases (KAS,e.g., KASI-KAS IV), Fatty acid thioesterase B (FATB, e.g., FATB1-FATB5,or other plastidial thioesterases), Fatty acid synthase (FAS), Fattyacid elongase (FAE, e.g., FAE1), Fatty acid thioesterase A (FatA), Fattyacid desaturase (Fad2, Fad3), plastidial G-3-P dehydrogenase (GPDH),glycerokinase (GK), stearoyl-acyl carrier protein desaturase(S-ACP-DES), and oleoyl-ACP hydrolase. In some particular embodiments,the gene can be an ortholog or a homolog of these genes in otheroil-producing plant species. Thus, the methods and compositions of theinvention may be used to disrupt a gene involved with fatty acidbiosynthesis with a CRISPR/Cas system where the single guide RNAcomprises sequences to target a fatty acid synthesis gene.

Malate dehydrogenase (MDH) catalyzes a reversibleNAD+-dependent-dehydrogenase reaction involved in central metabolism andredox homeostasis between organelle compartments in both plants andmammals. Plant tissues contain multiple isoforms of malate dehydrogenase(1-malate-NAD-oxidoreductase [MDH]; EC 1.1.1.37) that catalyze theinterconversion of malate and oxaloacetate (OAA) coupled to reduction oroxidation of the NAD pool. Notably, OAA is readily transported both intoand out of isolated plant mitochondria, in contrast to mammalianmitochondria, which are essentially impermeable to this organic acid.MDH-mutant plants exhibit either no obvious phenotype or slower growthrates and altered photorespiratory characteristics. See, e.g., Tomaz etal. (2010) Plant Physiol. 154(3):1143-1157, Goodman, M. M., Newton K. J.and Stuber, C. W. (1981) Proc. Nat. Acad. Sci. USA 78:1783-1785 orImsande, J. et al. (2001) J. Heredity 92:333-338 and U.S. PatentPublication No. 20090123626 describes the use of MDH antisense to reduceasparagine levels, which in turn lowers the level of acrylamide thataccumulates upon processing-associated heating of the plant and plantproducts. Thus, the methods and compositions of the invention may beused to disrupt a malate dehydrogenase (MDH) with a CRISPR/Cas systemwhere the single guide RNA comprises sequences to target a MDH gene.

The CRISPR/Cas system comprises an engineered sgRNA (or its equivalent)to target desired locations in a genome. Only 12 base pairs of thisguide RNA and 2 base pairs of the PAM sequence (14 base pairs total)appear to be critical for CRISPR/Cas activity. Assuming the CRISPR/Casconstruct is absolutely specific for these 14 base pairs, we wouldexpect such a construct to cleave approximately 20 times in the humangenome since a given 14 nucleotide sequence will occur approximatelyonce every 268,435,456 (4¹⁴) nucleotides, the haploid human genomecontains approximately 3,000,000,000 nucleotides, and the 14 nucleotidesequence can be on either of the two strands of the genome. This figurealso requires absolute specificity of recognition. So the actual numberof off target cleavage sites could be higher. The instant inventionprovides methods and compositions to increase the target size of theCRISPR/Cas system to increase its specificity. The Cas nucleasecomprises two nuclease domains, the HNH and RuvC-like, for cleaving thesense and the antisense strands of the target DNA, respectively. The Casnuclease can thus be engineered such that only one of the nucleasedomains is functional, thus creating a Cas nickase (see Jinek et al,ibid). Nickases can be generated by specific mutation of amino acids inthe catalytic domain of the enzyme, or by truncation of part or all ofthe domain such that it is no longer functional. Since Cas comprises twonuclease domains, this approach may be taken on either domain. A doublestrand break can be achieved in the target DNA by the use of two suchCas nickases. The nickases will each cleave one strand of the DNA andthe use of two will create a double strand break. Thus, specificity ofthe system is increased greatly by using two Cas nickase proteinsbecause the target length is increased from 12-14 nucleotides to 24-28nucleotides (not including the spacer in between the two subtargets).Using this approach, the expected number of perfectly matched off-targetsites in a human genome drops by a factor or 4¹⁴, from ˜20 (as describedabove) to ˜<10⁻⁷ (assuming a 28 base pair DNA recognized by theCRISPR/Cas nickase dimer).

General

Practice of the methods, as well as preparation and use of thecompositions disclosed herein employ, unless otherwise indicated,conventional techniques in molecular biology, biochemistry, chromatinstructure and analysis, computational chemistry, cell culture,recombinant DNA and related fields as are within the skill of the art.These techniques are fully explained in the literature. See, forexample, Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL, Secondedition, Cold Spring Harbor Laboratory Press, 1989 and Third edition,2001; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley& Sons, New York, 1987 and periodic updates; the series METHODS INENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATIN STRUCTURE ANDFUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS INENZYMOLOGY, Vol. 304, “Chromatin” (P. M. Wassarman and A. P. Wolffe,eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULARBIOLOGY, Vol. 119, “Chromatin Protocols” (P. B. Becker, ed.) HumanaPress, Totowa, 1999.

DEFINITIONS

The terms “nucleic acid,” “polynucleotide,” and “oligonucleotide” areused interchangeably and refer to a deoxyribonucleotide orribonucleotide polymer, in linear or circular conformation, and ineither single- or double-stranded form. For the purposes of the presentdisclosure, these terms are not to be construed as limiting with respectto the length of a polymer. The terms can encompass known analogues ofnatural nucleotides, as well as nucleotides that are modified in thebase, sugar and/or phosphate moieties (e.g., phosphorothioatebackbones). In general, an analogue of a particular nucleotide has thesame base-pairing specificity; i.e., an analogue of A will base-pairwith T.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably to refer to a polymer of amino acid residues. The termalso applies to amino acid polymers in which one or more amino acids arechemical analogues or modified derivatives of correspondingnaturally-occurring amino acids.

“Binding” refers to a sequence-specific, non-covalent interactionbetween macromolecules (e.g., between a protein and a nucleic acid orbetween two nucleic acids). Not all components of a binding interactionneed be sequence-specific (e.g., contacts with phosphate residues in aDNA backbone), as long as the interaction as a whole issequence-specific. Such interactions are generally characterized by adissociation constant (K_(d)) of 10⁻⁶ M⁻¹ or lower. “Affinity” refers tothe strength of binding: increased binding affinity being correlatedwith a lower K_(d).

The terms “chimeric RNA”, “chimeric guide RNA”, “guide RNA”, “singleguide RNA” and “synthetic guide RNA” are used interchangeably and referto the polynucleotide sequence comprising the guide sequence, the tracrsequence and the tracr mate sequence. The term “guide sequence” refersto the about 10-30 (10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30) base pair sequence within the guide RNAthat specifies the target site and may be used interchangeably with theterms “guide” or “spacer”. The term “tracr mate sequence” may also beused interchangeably with the term “direct repeat(s)”.

“Complementarity” refers to the ability of a nucleic acid to formhydrogen bond(s) with another nucleic acid sequence by eithertraditional Watson-Crick or other non-traditional types. A percentcomplementarity indicates the percentage of residues in a nucleic acidmolecule which can form hydrogen bonds (e.g., Watson-Crick base pairing)with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10being 50%, 60%, 70%, 80%, 90%, and 100% complementary). “Perfectlycomplementary” means that all the contiguous residues of a nucleic acidsequence will hydrogen bond with the same number of contiguous residuesin a second nucleic acid sequence. “Substantially complementary” as usedherein refers to a degree of complementarity that is at least 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%. 97%, 98%, 99%, or 100% over a region of 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30,35, 40, 45, 50, or more nucleotides, or refers to two nucleic acids thathybridize under stringent conditions. A “binding protein” is a proteinthat is able to bind to another molecule. A binding protein can bind to,for example, a DNA molecule (a DNA-binding protein), an RNA molecule (anRNA-binding protein) and/or a protein molecule (a protein-bindingprotein). In the case of a protein-binding protein, it can bind toitself (to form homodimers, homotrimers, etc.) and/or it can bind to oneor more molecules of a different protein or proteins. A binding proteincan have more than one type of binding activity. For example, zincfinger proteins have DNA-binding, RNA-binding and protein-bindingactivity.

A “zinc finger DNA binding protein” (or binding domain) is a protein, ora domain within a larger protein, that binds DNA in a sequence-specificmanner through one or more zinc fingers, which are regions of amino acidsequence within the binding domain whose structure is stabilized throughcoordination of a zinc ion. The term zinc finger DNA binding protein isoften abbreviated as zinc finger protein or ZFP.

A “TALE DNA binding domain” or “TALE” is a polypeptide comprising one ormore TALE repeat domains/units. The repeat domains are involved inbinding of the TALE to its cognate target DNA sequence. A single “repeatunit” (also referred to as a “repeat”) is typically 33-35 amino acids inlength and exhibits at least some sequence homology with other TALErepeat sequences within a naturally occurring TALE protein.

Zinc finger and TALE binding domains can be “engineered” to bind to apredetermined nucleotide sequence, for example via engineering (alteringone or more amino acids) of the recognition helix region of a naturallyoccurring zinc finger or TALE protein. Therefore, engineered DNA bindingproteins (zinc fingers or TALEs) are proteins that are non-naturallyoccurring. Non-limiting examples of methods for engineering DNA-bindingproteins are design and selection. A designed DNA binding protein is aprotein not occurring in nature whose design/composition resultsprincipally from rational criteria. Rational criteria for design includeapplication of substitution rules and computerized algorithms forprocessing information in a database storing information of existing ZFPand/or TALE designs and binding data. See, for example, U.S. Pat. Nos.8,586,526 6,140,081; 6,453,242; and 6,534,261; see also WO 98/53058; WO98/53059; WO 98/53060; WO 02/016536 and WO 03/016496.

A “selected” zinc finger protein or TALE is a protein not found innature whose production results primarily from an empirical process suchas phage display, interaction trap or hybrid selection. See e.g., U.S.Pat. Nos. 8,586,526; 5,789,538; 5,925,523; 6,007,988; 6,013,453;6,200,759; as well as WO 95/19431; WO 96/06166; WO 98/53057; WO98/54311; WO 00/27878; WO 01/60970 WO 01/88197; WO 02/099084.

“Recombination” refers to a process of exchange of genetic informationbetween two polynucleotides. For the purposes of this disclosure,“homologous recombination (HR)” refers to the specialized form of suchexchange that takes place, for example, during repair of double-strandbreaks in cells via homology-directed repair mechanisms. This processrequires nucleotide sequence homology, uses a “donor” molecule totemplate repair of a “target” molecule (i.e., the one that experiencedthe double-strand break), and is variously known as “non-crossover geneconversion” or “short tract gene conversion,” because it leads to thetransfer of genetic information from the donor to the target. Withoutwishing to be bound by any particular theory, such transfer can involvemismatch correction of heteroduplex DNA that forms between the brokentarget and the donor, and/or “synthesis-dependent strand annealing,” inwhich the donor is used to re-synthesize genetic information that willbecome part of the target, and/or related processes. Such specialized HRoften results in an alteration of the sequence of the target moleculesuch that part or all of the sequence of the donor polynucleotide isincorporated into the target polynucleotide.

In the methods of the disclosure, one or more targeted nucleases (e.g.,CRISPR/Cas) as described herein create a double-stranded break in thetarget sequence (e.g., cellular chromatin) at a predetermined site, anda “donor” polynucleotide, having homology to the nucleotide sequence inthe region of the break, can be introduced into the cell. The presenceof the double-stranded break has been shown to facilitate integration ofthe donor sequence. The donor sequence may be physically integrated or,alternatively, the donor polynucleotide is used as a template for repairof the break via homologous recombination, resulting in the introductionof all or part of the nucleotide sequence as in the donor into thecellular chromatin. Thus, a first sequence in cellular chromatin can bealtered and, in certain embodiments, can be converted into a sequencepresent in a donor polynucleotide. Thus, the use of the terms “replace”or “replacement” can be understood to represent replacement of onenucleotide sequence by another, (i.e., replacement of a sequence in theinformational sense), and does not necessarily require physical orchemical replacement of one polynucleotide by another.

In any of the methods described herein, additional CRISPR/Cas nucleasesand/or additional pairs of zinc-finger or TALEN proteins can be used foradditional double-stranded cleavage of additional target sites withinthe cell.

In certain embodiments of methods for targeted recombination and/orreplacement and/or alteration of a sequence in a region of interest incellular chromatin, a chromosomal sequence is altered by homologousrecombination with an exogenous “donor” nucleotide sequence. Suchhomologous recombination is stimulated by the presence of adouble-stranded break in cellular chromatin, if sequences homologous tothe region of the break are present.

In any of the methods described herein, the exogenous nucleotidesequence (the “donor sequence” or “transgene”) can contain sequencesthat are homologous, but not identical, to genomic sequences in theregion of interest, thereby stimulating homologous recombination toinsert a non-identical sequence in the region of interest. Thus, incertain embodiments, portions of the donor sequence that are homologousto sequences in the region of interest exhibit between about 80 to 99%(or any integer therebetween) sequence identity to the genomic sequencethat is replaced. In other embodiments, the homology between the donorand genomic sequence is higher than 99%, for example if only 1nucleotide differs as between donor and genomic sequences of over 100contiguous base pairs. In certain cases, a non-homologous portion of thedonor sequence can contain sequences not present in the region ofinterest, such that new sequences are introduced into the region ofinterest. In these instances, the non-homologous sequence is generallyflanked by sequences of 50-1,000 base pairs (or any integral valuetherebetween) or any number of base pairs greater than 1,000, that arehomologous or identical to sequences in the region of interest. In otherembodiments, the donor sequence is non-homologous to the first sequence,and is inserted into the genome by non-homologous recombinationmechanisms.

Any of the methods described herein can be used for partial or completeinactivation of one or more target sequences in a cell by targetedintegration of donor sequence that disrupts expression of the gene(s) ofinterest. Cell lines with partially or completely inactivated genes arealso provided.

Furthermore, the methods of targeted integration as described herein canalso be used to integrate one or more exogenous sequences. The exogenousnucleic acid sequence can comprise, for example, one or more genes orcDNA molecules, or any type of coding or non-coding sequence, as well asone or more control elements (e.g., promoters). In addition, theexogenous nucleic acid sequence may produce one or more RNA molecules(e.g., small hairpin RNAs (shRNAs), inhibitory RNAs (RNAis), microRNAs(miRNAs), etc.).

“Cleavage” refers to the breakage of the covalent backbone of a DNAmolecule. Cleavage can be initiated by a variety of methods including,but not limited to, enzymatic or chemical hydrolysis of a phosphodiesterbond. Both single-stranded cleavage and double-stranded cleavage arepossible, and double-stranded cleavage can occur as a result of twodistinct single-stranded cleavage events. DNA cleavage can result in theproduction of either blunt ends or staggered ends. In certainembodiments, fusion polypeptides are used for targeted double-strandedDNA cleavage.

A “cleavage half-domain” is a polypeptide sequence which, in conjunctionwith a second polypeptide (either identical or different) forms acomplex having cleavage activity (preferably double-strand cleavageactivity). The terms “first and second cleavage half-domains;” “+ and −cleavage half-domains” and “right and left cleavage half-domains” areused interchangeably to refer to pairs of cleavage half-domains thatdimerize.

An “engineered cleavage half-domain” is a cleavage half-domain that hasbeen modified so as to form obligate heterodimers with another cleavagehalf-domain (e.g., another engineered cleavage half-domain). See, also,U.S. Patent Publication Nos. 2005/0064474, 2007/0218528, 2008/0131962and 2011/0201055, incorporated herein by reference in their entireties.

The term “sequence” refers to a nucleotide sequence of any length, whichcan be DNA or RNA; can be linear, circular or branched and can be eithersingle-stranded or double stranded. The term “donor sequence” refers toa nucleotide sequence that is inserted into a genome. A donor sequencecan be of any length, for example between 2 and 10,000 nucleotides inlength (or any integer value therebetween or thereabove), preferablybetween about 100 and 1,000 nucleotides in length (or any integertherebetween), more preferably between about 200 and 500 nucleotides inlength.

A “homologous, non-identical sequence” refers to a first sequence whichshares a degree of sequence identity with a second sequence, but whosesequence is not identical to that of the second sequence. For example, apolynucleotide comprising the wild-type sequence of a mutant gene ishomologous and non-identical to the sequence of the mutant gene. Incertain embodiments, the degree of homology between the two sequences issufficient to allow homologous recombination therebetween, utilizingnormal cellular mechanisms. Two homologous non-identical sequences canbe any length and their degree of non-homology can be as small as asingle nucleotide (e.g., for correction of a genomic point mutation bytargeted homologous recombination) or as large as 10 or more kilobases(e.g., for insertion of a gene at a predetermined ectopic site in achromosome). Two polynucleotides comprising the homologous non-identicalsequences need not be the same length. For example, an exogenouspolynucleotide (i.e., donor polynucleotide) of between 20 and 10,000nucleotides or nucleotide pairs can be used.

Techniques for determining nucleic acid and amino acid sequence identityare known in the art. Typically, such techniques include determining thenucleotide sequence of the mRNA for a gene and/or determining the aminoacid sequence encoded thereby, and comparing these sequences to a secondnucleotide or amino acid sequence. Genomic sequences can also bedetermined and compared in this fashion. In general, identity refers toan exact nucleotide-to-nucleotide or amino acid-to-amino acidcorrespondence of two polynucleotides or polypeptide sequences,respectively. Two or more sequences (polynucleotide or amino acid) canbe compared by determining their percent identity. The percent identityof two sequences, whether nucleic acid or amino acid sequences, is thenumber of exact matches between two aligned sequences divided by thelength of the shorter sequences and multiplied by 100. An approximatealignment for nucleic acid sequences is provided by the local homologyalgorithm of Smith and Waterman, Advances in Applied Mathematics2:482-489 (1981). This algorithm can be applied to amino acid sequencesby using the scoring matrix developed by Dayhoff, Atlas of ProteinSequences and Structure, M. O. Dayhoff ed., 5 suppl. 3:353-358, NationalBiomedical Research Foundation, Washington, D.C., USA, and normalized byGribskov, Nucl. Acids Res. 14(6):6745-6763 (1986). An exemplaryimplementation of this algorithm to determine percent identity of asequence is provided by the Genetics Computer Group (Madison, Wis.) inthe “BestFit” utility application. Suitable programs for calculating thepercent identity or similarity between sequences are generally known inthe art, for example, another alignment program is BLAST, used withdefault parameters. For example, BLASTN and BLASTP can be used using thefollowing default parameters: genetic code=standard; filter=none;strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50sequences; sort by=HIGH SCORE; Databases=non-redundant,GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+Swissprotein+Spupdate+PIR. Details of these programs can be found on theinternet. With respect to sequences described herein, the range ofdesired degrees of sequence identity is approximately 80% to 100% andany integer value therebetween. Typically the percent identities betweensequences are at least 70-75%, preferably 80-82%, more preferably85-90%, even more preferably 92%, still more preferably 95%, and mostpreferably 98% sequence identity.

Alternatively, the degree of sequence similarity between polynucleotidescan be determined by hybridization of polynucleotides under conditionsthat allow formation of stable duplexes between homologous regions,followed by digestion with single-stranded-specific nuclease(s), andsize determination of the digested fragments. Two nucleic acid, or twopolypeptide sequences are substantially homologous to each other whenthe sequences exhibit at least about 70%-75%, preferably 80%-82%, morepreferably 85%-90%, even more preferably 92%, still more preferably 95%,and most preferably 98% sequence identity over a defined length of themolecules, as determined using the methods above. As used herein,substantially homologous also refers to sequences showing completeidentity to a specified DNA or polypeptide sequence. DNA sequences thatare substantially homologous can be identified in a Southernhybridization experiment under, for example, stringent conditions, asdefined for that particular system. Defining appropriate hybridizationconditions is known to those with skill of the art. See, e.g., Sambrooket al., supra; Nucleic Acid Hybridization: A Practical Approach, editorsB. D. Hames and S. J. Higgins, (1985) Oxford; Washington, D.C.; IRLPress).

Selective hybridization of two nucleic acid fragments can be determinedas follows. The degree of sequence identity between two nucleic acidmolecules affects the efficiency and strength of hybridization eventsbetween such molecules. A partially identical nucleic acid sequence willat least partially inhibit the hybridization of a completely identicalsequence to a target molecule. Inhibition of hybridization of thecompletely identical sequence can be assessed using hybridization assaysthat are well known in the art (e.g., Southern (DNA) blot, Northern(RNA) blot, solution hybridization, or the like, see Sambrook, et al.,Molecular Cloning: A Laboratory Manual, Second Edition, (1989) ColdSpring Harbor, N.Y.). Such assays can be conducted using varying degreesof selectivity, for example, using conditions varying from low to highstringency. If conditions of low stringency are employed, the absence ofnon-specific binding can be assessed using a secondary probe that lackseven a partial degree of sequence identity (for example, a probe havingless than about 30% sequence identity with the target molecule), suchthat, in the absence of non-specific binding events, the secondary probewill not hybridize to the target.

When utilizing a hybridization-based detection system, a nucleic acidprobe is chosen that is complementary to a reference nucleic acidsequence, and then by selection of appropriate conditions the probe andthe reference sequence selectively hybridize, or bind, to each other toform a duplex molecule. A nucleic acid molecule that is capable ofhybridizing selectively to a reference sequence under moderatelystringent hybridization conditions typically hybridizes under conditionsthat allow detection of a target nucleic acid sequence of at least about10-14 nucleotides in length having at least approximately 70% sequenceidentity with the sequence of the selected nucleic acid probe. Stringenthybridization conditions typically allow detection of target nucleicacid sequences of at least about 10-14 nucleotides in length having asequence identity of greater than about 90-95% with the sequence of theselected nucleic acid probe. Hybridization conditions useful forprobe/reference sequence hybridization, where the probe and referencesequence have a specific degree of sequence identity, can be determinedas is known in the art (see, for example, Nucleic Acid Hybridization: APractical Approach, editors B. D. Hames and S. J. Higgins, (1985)Oxford; Washington, D.C.; IRL Press).

Conditions for hybridization are well-known to those of skill in theart. Hybridization stringency refers to the degree to whichhybridization conditions disfavor the formation of hybrids containingmismatched nucleotides, with higher stringency correlated with a lowertolerance for mismatched hybrids. Factors that affect the stringency ofhybridization are well-known to those of skill in the art and include,but are not limited to, temperature, pH, ionic strength, andconcentration of organic solvents such as, for example, formamide anddimethylsulfoxide. As is known to those of skill in the art,hybridization stringency is increased by higher temperatures, lowerionic strength and lower solvent concentrations.

With respect to stringency conditions for hybridization, it is wellknown in the art that numerous equivalent conditions can be employed toestablish a particular stringency by varying, for example, the followingfactors: the length and nature of the probe sequences, base compositionof the various sequences, concentrations of salts and otherhybridization solution components, the presence or absence of blockingagents in the hybridization solutions (e.g., dextran sulfate, andpolyethylene glycol), hybridization reaction temperature and timeparameters, as well as, varying wash conditions. The selection of aparticular set of hybridization conditions is selected followingstandard methods in the art (see, for example, Sambrook, et al.,Molecular Cloning: A Laboratory Manual, Second Edition, (1989) ColdSpring Harbor, N.Y.).

“Chromatin” is the nucleoprotein structure comprising the cellulargenome. Cellular chromatin comprises nucleic acid, primarily DNA, andprotein, including histones and non-histone chromosomal proteins. Themajority of eukaryotic cellular chromatin exists in the form ofnucleosomes, wherein a nucleosome core comprises approximately 150 basepairs of DNA associated with an octamer comprising two each of histonesH2A, H2B, H3 and H4; and linker DNA (of variable length depending on theorganism) extends between nucleosome cores. A molecule of histone H1 isgenerally associated with the linker DNA. For the purposes of thepresent disclosure, the term “chromatin” is meant to encompass all typesof cellular nucleoprotein, both prokaryotic and eukaryotic. Cellularchromatin includes both chromosomal and episomal chromatin.

A “chromosome,” is a chromatin complex comprising all or a portion ofthe genome of a cell. The genome of a cell is often characterized by itskaryotype, which is the collection of all the chromosomes that comprisethe genome of the cell. The genome of a cell can comprise one or morechromosomes.

An “episome” is a replicating nucleic acid, nucleoprotein complex orother structure comprising a nucleic acid that is not part of thechromosomal karyotype of a cell. Examples of episomes include plasmidsand certain viral genomes.

An “accessible region” is a site in cellular chromatin in which a targetsite present in the nucleic acid can be bound by an exogenous moleculewhich recognizes the target site. Without wishing to be bound by anyparticular theory, it is believed that an accessible region is one thatis not packaged into a nucleosomal structure. The distinct structure ofan accessible region can often be detected by its sensitivity tochemical and enzymatic probes, for example, nucleases.

A “target site” or “target sequence” is a nucleic acid sequence thatdefines a portion of a nucleic acid to which a binding molecule willbind, provided sufficient conditions for binding exist.

An “exogenous” molecule is a molecule that is not normally present in acell, but can be introduced into a cell by one or more genetic,biochemical or other methods. “Normal presence in the cell” isdetermined with respect to the particular developmental stage andenvironmental conditions of the cell. Thus, for example, a molecule thatis present only during embryonic development of muscle is an exogenousmolecule with respect to an adult muscle cell. Similarly, a moleculeinduced by heat shock is an exogenous molecule with respect to anon-heat-shocked cell. An exogenous molecule can comprise, for example,a functioning version of a malfunctioning endogenous molecule or amalfunctioning version of a normally-functioning endogenous molecule.

An exogenous molecule can be, among other things, a small molecule, suchas is generated by a combinatorial chemistry process, or a macromoleculesuch as a protein, nucleic acid, carbohydrate, lipid, glycoprotein,lipoprotein, polysaccharide, any modified derivative of the abovemolecules, or any complex comprising one or more of the above molecules.Nucleic acids include DNA and RNA, can be single- or double-stranded;can be linear, branched or circular; and can be of any length. Nucleicacids include those capable of forming duplexes, as well astriplex-forming nucleic acids. See, for example, U.S. Pat. Nos.5,176,996 and 5,422,251. Proteins include, but are not limited to,DNA-binding proteins, transcription factors, chromatin remodelingfactors, methylated DNA binding proteins, polymerases, methylases,demethylases, acetylases, deacetylases, kinases, phosphatases,integrases, recombinases, ligases, topoisomerases, gyrases andhelicases. Thus, the term includes “transgenes” or “genes of interest”which are exogenous sequences introduced into a cell.

An exogenous molecule can be the same type of molecule as an endogenousmolecule, e.g., an exogenous protein or nucleic acid. For example, anexogenous nucleic acid can comprise an infecting viral genome, a plasmidor episome introduced into a cell, or a chromosome that is not normallypresent in the cell. Methods for the introduction of exogenous moleculesinto cells are known to those of skill in the art and include, but arenot limited to, lipid-mediated transfer (i.e., liposomes, includingneutral and cationic lipids), electroporation, direct injection, cellfusion, particle bombardment, calcium phosphate co-precipitation,DEAE-dextran-mediated transfer and viral vector-mediated transfer. Anexogenous molecule can also be the same type of molecule as anendogenous molecule but derived from a different species than the cellis derived from. For example, a human nucleic acid sequence may beintroduced into a cell line originally derived from a mouse or hamster.Methods for the introduction of exogenous molecules into plant cells areknown to those of skill in the art and include, but are not limited to,protoplast transformation, silicon carbide (e.g., WHISKERS™),Agrobacterium-mediated transformation, lipid-mediated transfer (i.e.,liposomes, including neutral and cationic lipids), electroporation,direct injection, cell fusion, particle bombardment (e.g., using a “genegun”), calcium phosphate co-precipitation, DEAE-dextran-mediatedtransfer and viral vector-mediated transfer.

By contrast, an “endogenous” molecule is one that is normally present ina particular cell at a particular developmental stage under particularenvironmental conditions. For example, an endogenous nucleic acid cancomprise a chromosome, the genome of a mitochondrion, chloroplast orother organelle, or a naturally-occurring episomal nucleic acid.Additional endogenous molecules can include proteins, for example,transcription factors and enzymes.

As used herein, the term “product of an exogenous nucleic acid” includesboth polynucleotide and polypeptide products, for example, transcriptionproducts (polynucleotides such as RNA) and translation products(polypeptides).

A “fusion” molecule is a molecule in which two or more subunit moleculesare linked, preferably covalently. The subunit molecules can be the samechemical type of molecule, or can be different chemical types ofmolecules. Examples of the first type of fusion molecule include, butare not limited to, fusion proteins (for example, a fusion between a ZFPor TALE DNA-binding domain and one or more activation domains) andfusion nucleic acids (for example, a nucleic acid encoding the fusionprotein described supra). Examples of the second type of fusion moleculeinclude, but are not limited to, a fusion between a triplex-formingnucleic acid and a polypeptide, and a fusion between a minor groovebinder and a nucleic acid. A “fusion polypeptide” is a polypeptidecomprising a polypeptide or portion (e.g., one or more domains) thereoffused or bonded to heterologous polypeptide. Examples of fusionpolypeptides include immunoadhesins which combine a portion of the Casprotein with an immunoglobulin sequence, and epitope taggedpolypeptides, which may comprise a Cas protein, for example, or portionthereof fused to a “tag polypeptide”. The tag polypeptide has enoughresidues to provide an epitope against which an antibody can be made,yet is short enough such that it does not interfere with nucleaseactivity of Cas. Suitable tag polypeptides generally have at least sixamino acid residues and usually between about 6-60 amino acid residues.

Expression of a fusion protein in a cell can result from delivery of thefusion protein to the cell or by delivery of a polynucleotide encodingthe fusion protein to a cell, wherein the polynucleotide is transcribed,and the transcript is translated, to generate the fusion protein.Trans-splicing, polypeptide cleavage and polypeptide ligation can alsobe involved in expression of a protein in a cell. Methods forpolynucleotide and polypeptide delivery to cells are presented elsewherein this disclosure.

A “gene,” for the purposes of the present disclosure, includes a DNAregion encoding a gene product (see infra), as well as all DNA regionswhich regulate the production of the gene product, whether or not suchregulatory sequences are adjacent to coding and/or transcribedsequences. Accordingly, a gene includes, but is not necessarily limitedto, promoter sequences, terminators, translational regulatory sequencessuch as ribosome binding sites and internal ribosome entry sites,enhancers, silencers, insulators, boundary elements, replicationorigins, matrix attachment sites and locus control regions. An“engineered gene” refers to a gene which has been altered in some mannersuch that it is non-identical with a wild type gene. Alterations can bein the form of targeted deletions, insertions and truncations. Anengineered gene can comprise coding sequences from two heterologousgenes or may comprise synthetic gene sequences. An engineered gene mayalso comprise changes in the coding sequence that are silent in theprotein sequence (e.g. codon optimization). An engineered gene can alsocomprise a gene with altered regulatory sequences.

“Gene expression” refers to the conversion of the information, containedin a gene, into a gene product. A gene product can be the directtranscriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisenseRNA, ribozyme, structural RNA or any other type of RNA) or a proteinproduced by translation of an mRNA. Gene products also include RNAswhich are modified, by processes such as capping, polyadenylation,methylation, and editing, and proteins modified by, for example,methylation, acetylation, phosphorylation, ubiquitination,ADP-ribosylation, myristilation, and glycosylation.

“Modulation” of gene expression refers to a change in the activity of agene. Modulation of expression can include, but is not limited to, geneactivation and gene repression. Genome editing (e.g., cleavage,alteration, inactivation, random mutation) can be used to modulateexpression. Gene inactivation refers to any reduction in gene expressionas compared to a cell that does not include a CRISPR/Cas system asdescribed herein. Thus, gene inactivation may be partial or complete.

“Plant” cells include, but are not limited to, cells of monocotyledonous(monocots) or dicotyledonous (dicots) plants. Non-limiting examples ofmonocots include cereal plants such as maize, rice, barley, oats, wheat,sorghum, rye, sugarcane, pineapple, onion, banana, and coconut.Non-limiting examples of dicots include tobacco, tomato, sunflower,cotton, sugarbeet, potato, lettuce, melon, soy, canola (rapeseed), andalfalfa. Plant cells may be from any part of the plant and/or from anystage of plant development.

A “region of interest” is any region of cellular chromatin, such as, forexample, a gene or a non-coding sequence within or adjacent to a gene,in which it is desirable to bind an exogenous molecule. Binding can befor the purposes of targeted DNA cleavage and/or targeted recombination.A region of interest can be present in a chromosome, an episome, anorganellar genome (e.g., mitochondrial, chloroplast), or an infectingviral genome, for example. A region of interest can be within the codingregion of a gene, within transcribed non-coding regions such as, forexample, leader sequences, trailer sequences or introns, or withinnon-transcribed regions, either upstream or downstream of the codingregion. A region of interest can be as small as a single nucleotide pairor up to 2,000 nucleotide pairs in length, or any integral value ofnucleotide pairs.

“Eukaryotic” cells include, but are not limited to, fungal cells (suchas yeast), plant cells, animal cells, mammalian cells and human cells(e.g., T-cells).

“Red Blood Cells” (RBCs), or erythrocytes, are terminally differentiatedcells derived from hematopoietic stem cells. They lack a nuclease andmost cellular organelles. RBCs contain hemoglobin to carry oxygen fromthe lungs to the peripheral tissues. In fact, 33% of an individual RBCis hemoglobin. They also carry CO2 produced by cells during metabolismout of the tissues and back to the lungs for release during exhale. RBCsare produced in the bone marrow in response to blood hypoxia which ismediated by release of erythropoietin (EPO) by the kidney. EPO causes anincrease in the number of proerythroblasts and shortens the timerequired for full RBC maturation. After approximately 120 days, sincethe RBC do not contain a nucleus or any other regenerative capabilities,the cells are removed from circulation by either the phagocyticactivities of macrophages in the liver, spleen and lymph nodes (˜90%) orby hemolysis in the plasma (˜10%). Following macrophage engulfment,chemical components of the RBC are broken down within vacuoles of themacrophages due to the action of lysosomal enzymes.

“Secretory tissues” are those tissues in an animal that secrete productsout of the individual cell into a lumen of some type which are typicallyderived from epithelium. Examples of secretory tissues that arelocalized to the gastrointestinal tract include the cells that line thegut, the pancreas, and the gallbladder. Other secretory tissues includethe liver, tissues associated with the eye and mucous membranes such assalivary glands, mammary glands, the prostate gland, the pituitary glandand other members of the endocrine system. Additionally, secretorytissues include individual cells of a tissue type which are capable ofsecretion.

The terms “operative linkage” and “operatively linked” (or “operablylinked”) are used interchangeably with reference to a juxtaposition oftwo or more components (such as sequence elements), in which thecomponents are arranged such that both components function normally andallow the possibility that at least one of the components can mediate afunction that is exerted upon at least one of the other components. Byway of illustration, a transcriptional regulatory sequence, such as apromoter, is operatively linked to a coding sequence if thetranscriptional regulatory sequence controls the level of transcriptionof the coding sequence in response to the presence or absence of one ormore transcriptional regulatory factors. A transcriptional regulatorysequence is generally operatively linked in cis with a coding sequence,but need not be directly adjacent to it. For example, an enhancer is atranscriptional regulatory sequence that is operatively linked to acoding sequence, even though they are not contiguous.

With respect to fusion polypeptides, the term “operatively linked” canrefer to the fact that each of the components performs the same functionin linkage to the other component as it would if it were not so linked.For example, with respect to a fusion polypeptide in which a CasDNA-binding domain is fused to an activation domain, the Cas DNA-bindingdomain and the activation domain are in operative linkage if, in thefusion polypeptide, the Cas DNA-binding domain portion is able to bindits target site and/or its binding site, while the activation domain isable to up-regulate gene expression. When a fusion polypeptide in whicha Cas DNA-binding domain is fused to a cleavage domain, the CasDNA-binding domain and the cleavage domain are in operative linkage if,in the fusion polypeptide, the Cas DNA-binding domain portion is able tobind its target site and/or its binding site, while the cleavage domainis able to cleave DNA in the vicinity of the target site.

A “functional fragment” of a protein, polypeptide or nucleic acid is aprotein, polypeptide or nucleic acid whose sequence is not identical tothe full-length protein, polypeptide or nucleic acid, yet retains thesame function as the full-length protein, polypeptide or nucleic acid. Afunctional fragment can possess more, fewer, or the same number ofresidues as the corresponding native molecule, and/or can contain one ormore amino acid or nucleotide substitutions. Methods for determining thefunction of a nucleic acid (e.g., coding function, ability to hybridizeto another nucleic acid) are well-known in the art. Similarly, methodsfor determining protein function are well-known. For example, theDNA-binding function of a polypeptide can be determined, for example, byfilter-binding, electrophoretic mobility-shift, or immunoprecipitationassays. DNA cleavage can be assayed by gel electrophoresis. See Ausubelet al., supra. The ability of a protein to interact with another proteincan be determined, for example, by co-immunoprecipitation, two-hybridassays or complementation, both genetic and biochemical. See, forexample, Fields et al. (1989) Nature 340:245-246; U.S. Pat. No.5,585,245 and PCT WO 98/44350.

A “vector” is capable of transferring gene sequences to target cells.Typically, “vector construct,” “expression vector,” and “gene transfervector,” mean any nucleic acid construct capable of directing theexpression of a gene of interest and which can transfer gene sequencesto target cells. Thus, the term includes cloning, and expressionvehicles, as well as integrating vectors.

A “reporter gene” or “reporter sequence” refers to any sequence thatproduces a protein product that is easily measured, preferably althoughnot necessarily in a routine assay. Suitable reporter genes include, butare not limited to, sequences encoding proteins that mediate antibioticresistance (e.g., ampicillin resistance, neomycin resistance, G418resistance, puromycin resistance), sequences encoding colored orfluorescent or luminescent proteins (e.g., green fluorescent protein,enhanced green fluorescent protein, red fluorescent protein,luciferase), and proteins which mediate enhanced cell growth and/or geneamplification (e.g., dihydrofolate reductase). Epitope tags include, forexample, one or more copies of FLAG, His, myc, Tap, HA or any detectableamino acid sequence. “Expression tags” include sequences that encodereporters that may be operably linked to a desired gene sequence inorder to monitor expression of the gene of interest.

The terms “subject” and “patient” are used interchangeably and refer tomammals such as human patients and non-human primates, as well asexperimental animals such as rabbits, dogs, cats, rats, mice, and otheranimals. Accordingly, the term “subject” or “patient” as used hereinmeans any mammalian patient or subject to which the or stem cells of theinvention can be administered. Subjects of the present invention includethose that have been exposed to one or more chemical toxins, including,for example, a nerve toxin.

The CRISPR/Cas System

Compelling evidence has recently emerged for the existence of anRNA-mediated genome defense pathway in archaea and many bacteria thathas been hypothesized to parallel the eukaryotic RNAi pathway (forreviews, see Godde and Bickerton, 2006. J. Mol. Evol. 62: 718-729;Lillestol et al., 2006. Archaea 2: 59-72; Makarova et al., 2006. Biol.Direct 1: 7.; Sorek et al., 2008. Nat. Rev. Microbiol. 6: 181-186).Known as the CRISPR-Cas system or prokaryotic RNAi (pRNAi), the pathwayis proposed to arise from two evolutionarily and often physically linkedgene loci: the CRISPR (clustered regularly interspaced short palindromicrepeats) locus, which encodes RNA components of the system, and the cas(CRISPR-associated) locus, which encodes proteins (Jansen et al., 2002.Mol. Microbiol. 43: 1565-1575; Makarova et al., 2002. Nucleic Acids Res.30: 482-496; Makarova et al., 2006. Biol. Direct 1: 7; Haft et al.,2005. PLoS Comput. Biol. 1: e60). CRISPR loci in microbial hosts containa combination of CRISPR-associated (Cas) genes as well as non-coding RNAelements capable of programming the specificity of the CRISPR-mediatednucleic acid cleavage. The individual Cas proteins do not sharesignificant sequence similarity with protein components of theeukaryotic RNAi machinery, but have analogous predicted functions (e.g.,RNA binding, nuclease, helicase, etc.) (Makarova et al., 2006. Biol.Direct 1: 7). The CRISPR-associated (cas) genes are often associatedwith CRISPR repeat-spacer arrays. More than forty different Cas proteinfamilies have been described. Of these protein families, Cas1 appears tobe ubiquitous among different CRISPR/Cas systems. Particularcombinations of cas genes and repeat structures have been used to define8 CRISPR subtypes (Ecoli, Ypest, Nmeni, Dvulg, Tneap, Hmari, Apern, andMtube), some of which are associated with an additional gene moduleencoding repeat-associated mysterious proteins (RAMPs). More than oneCRISPR subtype may occur in a single genome. The sporadic distributionof the CRISPR/Cas subtypes suggests that the system is subject tohorizontal gene transfer during microbial evolution.

The Type II CRISPR, initially described in S. pyogenes, is one of themost well characterized systems and carries out targeted DNAdouble-strand break in four sequential steps. First, two non-coding RNA,the pre-crRNA array and tracrRNA, are transcribed from the CRISPR locus.Second, tracrRNA hybridizes to the repeat regions of the pre-crRNA andmediates the processing of pre-crRNA into mature crRNAs containingindividual spacer sequences where processing occurs by a doublestrand-specific RNase III in the presence of the Cas9 protein. Third,the mature crRNA:tracrRNA complex directs Cas9 to the target DNA viaWatson-Crick base-pairing between the spacer on the crRNA and theprotospacer on the target DNA next to the protospacer adjacent motif(PAM), an additional requirement for target recognition. In addition,the tracrRNA must also be present as it base pairs with the crRNA at its3′ end, and this association triggers Cas9 activity. Finally, Cas9mediates cleavage of target DNA to create a double-stranded break withinthe protospacer. Activity of the CRISPR/Cas system comprises of threesteps: (i) insertion of alien DNA sequences into the CRISPR array toprevent future attacks, in a process called ‘adaptation,’ (ii)expression of the relevant proteins, as well as expression andprocessing of the array, followed by (iii) RNA-mediated interferencewith the alien nucleic acid. Thus, in the bacterial cell, several of theso-called ‘Cas’ proteins are involved with the natural function of theCRISPR/Cas system.

Type II CRISPR systems have been found in many different bacteria. BLASTsearches on publically available genomes by Fonfara et al ((2013) NucAcid Res 42(4):2377-2590) found Cas9 orthologs in 347 species ofbacteria. Additionally, this group demonstrated in vitro CRISPR/Cascleavage of a DNA target using Cas9 orthologs from S. pyogenes, S.mutans, S. therophilus, C. jejuni, N. meningitides, P. multocida and F.novicida. Thus, the term “Cas9” refers to an RNA guided DNA nucleasecomprising a DNA binding domain and two nuclease domains, where the geneencoding the Cas9 may be derived from any suitable bacteria.

The Cas9 protein has at least two nuclease domains: one nuclease domainis similar to a HNH endonuclease, while the other resembles a Ruvendonuclease domain. The HNH-type domain appears to be responsible forcleaving the DNA strand that is complementary to the crRNA while the Ruvdomain cleaves the non-complementary strand. The Cas 9 nuclease can beengineered such that only one of the nuclease domains is functional,creating a Cas nickase (see Jinek et al, ibid). Nickases can begenerated by specific mutation of amino acids in the catalytic domain ofthe enzyme, or by truncation of part or all of the domain such that itis no longer functional. Since Cas 9 comprises two nuclease domains,this approach may be taken on either domain. A double strand break canbe achieved in the target DNA by the use of two such Cas 9 nickases. Thenickases will each cleave one strand of the DNA and the use of two willcreate a double strand break.

The primary products of the CRISPR loci appear to be short RNAs thatcontain the invader targeting sequences, and are termed guide RNAs orprokaryotic silencing RNAs (psiRNAs) based on their hypothesized role inthe pathway (Makarova et al., 2006. Biol. Direct 1: 7; Hale et al.,2008. RNA, 14: 2572-2579). RNA analysis indicates that CRISPR locustranscripts are cleaved within the repeat sequences to release ^(˜)60-to 70-nt RNA intermediates that contain individual invader targetingsequences and flanking repeat fragments (Tang et al. 2002. Proc. Natl.Acad. Sci. 99: 7536-7541; Tang et al., 2005. Mol. Microbiol. 55:469-481; Lillestol et al. 2006. Archaea 2: 59-72; Brouns et al. 2008.Science 321: 960-964; Hale et al, 2008. RNA, 14: 2572-2579). In thearchaeon Pyrococcus furiosus, these intermediate RNAs are furtherprocessed to abundant, stable ^(˜)35- to 45-nt mature psiRNAs (Hale etal. 2008. RNA, 14: 2572-2579). The requirement of the crRNA-tracrRNAcomplex can be avoided by use of an engineered “single-guide RNA”(sgRNA) that comprises the hairpin normally formed by the annealing ofthe crRNA and the tracrRNA (see Jinek et al (2012) Science 337:816 andCong et al (2013) Sciencexpress/10.1126/science.1231143). In S.pyrogenes, the engineered tracrRNA:crRNA fusion, or the sgRNA, guidesCas9 to cleave the target DNA when a double strand RNA:DNA heterodimerforms between the Cas associated RNAs and the target DNA. This systemcomprising the Cas9 protein and an engineered sgRNA containing a PAMsequence has been used for RNA guided genome editing (see Ramalingamibid) and has been useful for zebrafish embryo genomic editing in vivo(see Hwang et al (2013) Nature Biotechnology 31 (3):227) with editingefficiencies similar to ZFNs and TALENs.

Other Cas Proteins

“Cas1” polypeptide refers to CRISPRassociated (Cas) protein1. Cas1(COG1518 in the Clusters of Orthologous Group of proteins classificationsystem) is the best marker of the CRISPR-associated systems (CASS).Based on phylogenetic comparisons, seven distinct versions of theCRISPR-associated immune system have been identified (CASS1-7).

Cas1 polypeptide used in the methods described herein can be any Cas1polypeptide present in any prokaryote. In certain embodiments, a Cas1polypeptide is a Cas1 polypeptide of an archaeal microorganism. Incertain embodiments, a Cas1 polypeptide is a Cas1 polypeptide of aEuryarchaeota microorganism. In certain embodiments, a Cas1 polypeptideis a Cas1 polypeptide of a Crenarchaeota microorganism. In certainembodiments, a Cas1 polypeptide is a Cas1 polypeptide of a bacterium. Incertain embodiments, a Cas1 polypeptide is a Cas1 polypeptide of a gramnegative or gram positive bacteria. In certain embodiments, a Cas1polypeptide is a Cas1 polypeptide of Pseudomonas aeruginosa. In certainembodiments, a Cas1 polypeptide is a Cas1 polypeptide of Aquifexaeolicus. In certain embodiments, a Cas1 polypeptide is a Cas1polypeptide that is a member of one of CASS1-7. In certain embodiments,Cas1 polypeptide is a Cas1 polypeptide that is a member of CASS3. Incertain embodiments, a Cas1 polypeptide is a Cas1 polypeptide that is amember of CASS7. In certain embodiments, a Cas1 polypeptide is a Cas1polypeptide that is a member of CASS3 or CASS7.

In some embodiments, a Cas1 polypeptide is encoded by a nucleotidesequence provided in GenBank at, e.g., GeneID number: 2781520, 1006874,9001811, 947228, 3169280, 2650014, 1175302, 3993120, 4380485, 906625,3165126, 905808, 1454460, 1445886, 1485099, 4274010, 888506, 3169526,997745, 897836, or 1193018 and/or an amino acid sequence exhibitinghomology (e.g., greater than 80%, 90 to 99% including 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99%) to the amino acids encoded by thesepolynucleotides and which polypeptides function as Cas1 polypeptides.

Cas6 is another Cas polypeptide, and the endoribonuclease activity isreferred to herein as Cas6 endoribonuclease activity. Non-limitingexamples of suitable Cas6 polypeptides are depicted at Genbank AccessionNo. AAL81255. A Cas6 polypeptide may be enriched, isolated, or purifiedfrom a microbe having a CRISPR locus and the cas (CRISPR-associated)locus, such as, but not limited to, Pyrococcus furiosus, or may beproduced using recombinant techniques, or chemically or enzymaticallysynthesized using routine methods. In some aspects, a Cas6 polypeptidemay be enriched, isolated, or purified from a microbe that does not haveCRISPR loci. A Cas6 polypeptide may include a GhGxxxxxGhG (SEQ IDNO:216) motif (where “h” indicates a hydrophobic amino acid) near theC-terminus. An Arg or Lys may be, and often is, found within the centralstretch of 5 amino acids. A Cas6 polypeptide contains at least oneresidue that may play a role in catalysis, or conservative substitutionthereof. A Cas6 polypeptide may contain other residues which may alsoplay a role in catalysis, or conservative substitution thereof. Theresidue(s) expected to play a role in catalysis may be located near theG-rich loop that contains the Cas6 signature motif in the 3D structureof the protein. Cas6 polypeptides may include domains present in theTIGRFAM database at accession numbers TIGR01877 and PF01881. The TIGRFAMdatabase includes families of polypeptides for which function isconserved (Haft et al., Nucl. Acids Res., 2003, 31:371-373, Bateman andHaft, 2002, Briefings Bioinformatics, 3:236-245, and Haft et al., 2005,PLoS Computational Biol., 1(6):e60).

Other examples of Cas6 polypeptides provided herein include thosepresent in prokaryotic microbes having a CRISPR locus and a cas locus.Cas6 polypeptides can be easily identified in any microbe that includesa CRISPR locus. A coding region encoding a Cas6 polypeptide is typicallyin a cas locus located in close proximity to a CRISPR locus. Haft et al.(2005, PLoS Computational Biol., 1(6):e60) review the Cas proteinfamily, and created rules for the identification of specific subtypes ofthe CRISPR/Cas system. Haft et al describe the coding region encodingCas6 polypeptides as being found in association with at least fourseparate CRISPR/Cas subtypes (Tneap, Hmari, Apern, and Mtube), and astypically being the cas coding region located most distal to the CRISPRlocus. Cas6 polypeptides may be identified using the resources availableat the JCVI Comprehensive Microbial Resource. Thus, Cas6 polypeptidesthat are useful in the methods described herein can be identified by theskilled person using routine methods.

Examples of prokaryotic microbes with known whole genomic sequencescontaining coding regions expected to encode a Cas6 polypeptide includeThermotoga maritima MSB8, Campylobacter fetus subsp. fetus 82-40,Fusobacterium nucleatum ATCC 25586, Streptococcus thermophilus LMG18311, Thermoanaerobacter tengcongensis MB4(T), Moorella thermoaceticaATCC 39073, Desulfitobacterium hafniense Y51, Clostridium tetani E88,Clostridium perfringens SM101, Clostridium difficile QCD-32g58,Clostridium botulinum Hall A Sanger, Clostridium botulinum F Langeland,Clostridium botulinum B1 strain Okra, Clostridium botulinum A3 strainLoch Maree, Clostridium botulinum A Hall, Clostridium botulinum A ATCC19397, Carboxydothermus hydrogenoformans Z-2901, Staphylococcusepidermidis RP62A, Thermus thermophilus HB8, Thermus thermophilus HB27,Nostoc sp. PCC 7120, Anabaena variabilis ATCC 29413, Synechococccus sp.OS Type B prime, Synechococccus sp. OS Type A, Porphyromonas gingivalisW83, Bacteroides fragilis YCH46, Bacteroides fragilis NCTC9343, Aquifexaeolicus VF5, Rubrobacter xylanophilus DSM 9941, Mycobacteriumtuberculosis H37Rv (lab strain), Mycobacterium tuberculosis CDC1551,Mycobacterium bovis subsp. bovis AF2122/97, Frankia alni ACN14a,Thermoplasma volcanium GSS1, Picrophilus torridus DSM 9790, Thermococcuskodakarensis KOD1, Pyrococcus horikoshii shinkaj OT3, Pyrococcusfuriosus DSM 3638, Pyrococcus abyssi GES, Methanosarcina barkeri fusaro,Methanosarcina acetivorans C2A, Methanococcoides burtonii DSM 6242,Methanococcus jannaschii DSM2661, Methanobacterium thermoautotrophicumdelta H, Haloarcula marismortui ATCC 43049, Archaeoglobus fulgidusDSM4304, Pyrobaculum aerophilum 1M2, Sulfolobus tokodaii strain 7,Sulfolobus solfataricus P2, Sulfolobus acidocaldarius DSM 639, Aeropyrumpernix K1. Other examples of Cas6 polypeptides are known to the skilledperson, see, for instance, members of the COG1583 group of polypeptides(available at the Clusters of Orthologous Groups of proteins (COGs) webpage through the National Center for Biotechnology Information internetsite, see also Tatusov et al., (1997), Science, 278:631-637, and Tatusovet al. (2003), BMC Bioinformatics, 4(1):41), members of the InterProfamily having accession number IPRO10156, Makarova et al., (2002), Nuc.Acids Res., 30:482-496 and Haft et al. (2005), PLoS Comput. Biol.,1(6):e60, 474-483).

In certain embodiments, Cas protein may be a “functional derivative” ofa naturally occurring Cas protein. A “functional derivative” of a nativesequence polypeptide is a compound having a qualitative biologicalproperty in common with a native sequence polypeptide. “Functionalderivatives” include, but are not limited to, fragments of a nativesequence and derivatives of a native sequence polypeptide and itsfragments, provided that they have a biological activity in common witha corresponding native sequence polypeptide. A biological activitycontemplated herein is the ability of the functional derivative tohydrolyze a DNA substrate into fragments. The term “derivative”encompasses both amino acid sequence variants of polypeptide, covalentmodifications, and fusions thereof. In some aspects, a functionalderivative may comprise a single biological property of a naturallyoccurring Cas protein. In other aspects, a function derivative maycomprise a subset of biological properties of a naturally occurring Casprotein.

“Cas1 polypeptide” encompasses a full-length Cas polypeptide, anenzymatically active fragment of a Cas polypeptide, and enzymaticallyactive derivatives of a Cas polypeptide or fragment thereof. Suitablederivatives of a Cas polypeptide or a fragment thereof include but arenot limited to mutants, fusions, covalent modifications of Cas proteinor a fragment thereof. Cas protein which includes Cas protein or afragment thereof, as well as derivatives of Cas protein or a fragmentthereof, may be obtainable from a cell or synthesized chemically or by acombination of these two procedures. The cell may be a cell thatnaturally produces Cas protein, or a cell that naturally produces Casprotein and is genetically engineered to produce the endogenous Casprotein at a higher expression level or to produce a Cas protein from anexogenously introduced nucleic acid, which nucleic acid encodes a Casthat is same or different from the endogenous Cas. In some case, thecell does not naturally produce Cas protein and is geneticallyengineered to produce a Cas protein.

The CRISPR/Cas system can also be used to inhibit gene expression. Leiet al (see, (2013) Cell, 152, (5): 1173-1183) have shown that acatalytically dead Cas9 lacking endonuclease activity, when coexpressedwith a guide RNA, generates a DNA recognition complex that canspecifically interfere with transcriptional elongation, RNA polymerasebinding, or transcription factor binding. This system, called CRISPRinterference (CRISPRi), can efficiently repress expression of targetedgenes.

The Cas proteins of the invention may be mutated to alter functionality.Exemplary selection methods, including phage display and two-hybridsystems, are disclosed in U.S. Pat. Nos. 5,789,538; 5,925,523;6,007,988; 6,013,453; 6,410,248; 6,140,466; 6,200,759; and 6,242,568; aswell as WO 98/37186; WO 98/53057; WO 00/27878; WO 01/88197 and GB2,338,237. In addition, enhancement of binding specificity for zincfinger binding domains has been described, for example, in co-owned WO02/077227.

Selection of target sites; DNA-binding domains and methods for designand construction of fusion proteins (and polynucleotides encoding same)are known to those of skill in the art and described in detail in U.S.Pat. Nos. 8,586,526; 6,140,081; 5,789,538; 6,453,242; 6,534,261;5,925,523; 6,007,988; 6,013,453; 6,200,759; WO 95/19431; WO 96/06166; WO98/53057; WO 98/54311; WO 00/27878; WO 01/60970 WO 01/88197; WO02/099084; WO 98/53058; WO 98/53059; WO 98/53060; WO 02/016536 and WO03/016496.

B. Functional Domains

The systems described herein can include any suitable functional domainthat associates with the single guide RNA to act on the target gene.Non-limiting examples of functional domains include transcriptionalactivation domains, transcriptional repression domains and/or nucleasedomains. See, e.g., U.S. Pat. Nos. 6,534,261; and 8,409,861.

In certain embodiments, the functional domain comprises one or morecleavage domains. The functional domain (e.g., cleavage domain) may beheterologous to the DNA-binding domain, for example a functional domainthat is heterologous to the single-guide RNA of the CRISPR/Cas system.Heterologous cleavage domains can be obtained from any endonuclease orexonuclease. Exemplary endonucleases from which a cleavage domain can bederived include, but are not limited to, restriction endonucleases andhoming endonucleases. See, for example, 2002-2003 Catalogue, New EnglandBiolabs, Beverly, Mass.; and Belfort et al. (1997) Nucleic Acids Res.25:3379-3388. Additional enzymes which cleave DNA are known (e.g., S1Nuclease; mung bean nuclease; pancreatic DNase I; micrococcal nuclease;yeast HO endonuclease; see also Linn et al. (eds.) Nucleases, ColdSpring Harbor Laboratory Press, 1993). One or more of these enzymes (orfunctional fragments thereof) can be used as a source of cleavagedomains and cleavage half-domains. In some embodiments, a Cas proteinmay be linked to a heterologous nuclease domain. In some aspects, theCas protein is a Cas9 protein devoid of nuclease activity linked to aFokI nuclease domain such that double strand cleavage is dependent ondimerization of the FokI nuclease domains.

Similarly, a cleavage half-domain can be derived from any nuclease orportion thereof, as set forth above, that requires dimerization forcleavage activity. In general, two fusion proteins are required forcleavage if the fusion proteins comprise cleavage half-domains.Alternatively, a single protein comprising two cleavage half-domains canbe used. The two cleavage half-domains can be derived from the sameendonuclease (or functional fragments thereof), or each cleavagehalf-domain can be derived from a different endonuclease (or functionalfragments thereof). In addition, the target sites for the two fusionproteins are preferably disposed, with respect to each other, such thatbinding of the two fusion proteins to their respective target sitesplaces the cleavage half-domains in a spatial orientation to each otherthat allows the cleavage half-domains to form a functional cleavagedomain, e.g., by dimerizing. Thus, in certain embodiments, the nearedges of the target sites are separated by 5-8 nucleotides or by 15-18nucleotides. However any integral number of nucleotides or nucleotidepairs can intervene between two target sites (e.g., from 2 to 50nucleotide pairs or more). In general, the site of cleavage lies betweenthe target sites.

Restriction endonucleases (restriction enzymes) are present in manyspecies and are capable of sequence-specific binding to DNA (at arecognition site), and cleaving DNA at or near the site of binding.Certain restriction enzymes (e.g., Type IIS) cleave DNA at sites removedfrom the recognition site and have separable binding and cleavagedomains. For example, the Type IIS enzyme Fok I catalyzesdouble-stranded cleavage of DNA, at 9 nucleotides from its recognitionsite on one strand and 13 nucleotides from its recognition site on theother. See, for example, U.S. Pat. Nos. 5,356,802; 5,436,150 and5,487,994; as well as Li et al. (1992) Proc. Natl. Acad. Sci. USA89:4275-4279; Li et al. (1993) Proc. Natl. Acad. Sci. USA 90:2764-2768;Kim et al. (1994a) Proc. Natl. Acad. Sci. USA 91:883-887; Kim et al.(1994b) J. Biol. Chem. 269:31,978-31,982. Thus, in one embodiment,fusion proteins comprise the cleavage domain (or cleavage half-domain)from at least one Type IIS restriction enzyme and one or more zincfinger binding domains, which may or may not be engineered.

An exemplary Type IIS restriction enzyme, whose cleavage domain isseparable from the binding domain, is Fok I. This particular enzyme isactive as a dimer. Bitinaite et al. (1998) Proc. Natl. Acad. Sci. USA95: 10,570-10,575. Accordingly, for the purposes of the presentdisclosure, the portion of the Fok I enzyme used in the disclosed fusionproteins is considered a cleavage half-domain. Thus, for targeteddouble-stranded cleavage and/or targeted replacement of cellularsequences using zinc finger-Fok I fusions, two fusion proteins, eachcomprising a FokI cleavage half-domain, can be used to reconstitute acatalytically active cleavage domain. Alternatively, a singlepolypeptide molecule containing a DNA binding domain and two Fok Icleavage half-domains can also be used.

A cleavage domain or cleavage half-domain can be any portion of aprotein that retains cleavage activity, or that retains the ability tomultimerize (e.g., dimerize) to form a functional cleavage domain.

Exemplary Type IIS restriction enzymes are described in InternationalPublication WO 07/014,275, incorporated herein in its entirety.Additional restriction enzymes also contain separable binding andcleavage domains, and these are contemplated by the present disclosure.See, for example, Roberts et al. (2003) Nucleic Acids Res. 31:418-420.

In certain embodiments, the cleavage domain comprises one or moreengineered cleavage half-domain (also referred to as dimerization domainmutants) that minimize or prevent homodimerization, as described, forexample, in U.S. Pat. Nos. 7,914,796; 8,034,598; and 8,623,618, thedisclosures of all of which are incorporated by reference in theirentireties herein. Amino acid residues at positions 446, 447, 479, 483,484, 486, 487, 490, 491, 496, 498, 499, 500, 531, 534, 537, and 538 ofFok I are all targets for influencing dimerization of the Fok I cleavagehalf-domains.

Exemplary engineered cleavage half-domains of Fok I that form obligateheterodimers include a pair in which a first cleavage half-domainincludes mutations at amino acid residues at positions 490 and 538 ofFok I and a second cleavage half-domain includes mutations at amino acidresidues 486 and 499.

Thus, in one embodiment, a mutation at 490 replaces Glu (E) with Lys(K); the mutation at 538 replaces Iso (I) with Lys (K); the mutation at486 replaced Gln (Q) with Glu (E); and the mutation at position 499replaces Iso (I) with Lys (K). Specifically, the engineered cleavagehalf-domains described herein were prepared by mutating positions 490(E→K) and 538 (I→K) in one cleavage half-domain to produce an engineeredcleavage half-domain designated “E490K:I538K” and by mutating positions486 (Q→E) and 499 (I→L) in another cleavage half-domain to produce anengineered cleavage half-domain designated “Q486E:I499L”. The engineeredcleavage half-domains described herein are obligate heterodimer mutantsin which aberrant cleavage is minimized or abolished. In certainembodiments, the engineered cleavage half-domain comprises mutations atpositions 486, 499 and 496 (numbered relative to wild-type FokI), forinstance mutations that replace the wild type Gln (Q) residue atposition 486 with a Glu (E) residue, the wild type Iso (I) residue atposition 499 with a Leu (L) residue and the wild-type Asn (N) residue atposition 496 with an Asp (D) or Glu (E) residue (also referred to as a“ELD” and “ELE” domains, respectively). In other embodiments, theengineered cleavage half-domain comprises mutations at positions 490,538 and 537 (numbered relative to wild-type FokI), for instancemutations that replace the wild type Glu (E) residue at position 490with a Lys (K) residue, the wild type Iso (I) residue at position 538with a Lys (K) residue, and the wild-type His (H) residue at position537 with a Lys (K) residue or a Arg (R) residue (also referred to as“KKK” and “KKR” domains, respectively). In other embodiments, theengineered cleavage half-domain comprises mutations at positions 490 and537 (numbered relative to wild-type FokI), for instance mutations thatreplace the wild type Glu (E) residue at position 490 with a Lys (K)residue and the wild-type His (H) residue at position 537 with a Lys (K)residue or a Arg (R) residue (also referred to as “KIK” and “KIR”domains, respectively). (See US Patent Publication No. 20110201055,incorporated by reference herein). Engineered cleavage half-domainsdescribed herein can be prepared using any suitable method, for example,by site-directed mutagenesis of wild-type cleavage half-domains (Fok I).

Alternatively, nucleases may be assembled in vivo at the nucleic acidtarget site using so-called “split-enzyme” technology (see, e.g., U.S.Patent Publication No. 20090068164). Components of such split enzymesmay be expressed either on separate expression constructs, or can belinked in one open reading frame where the individual components areseparated, for example, by a self-cleaving 2A peptide or IRES sequence.Components may be individual zinc finger binding domains or domains of ameganuclease nucleic acid binding domain.

Nucleases can be screened for activity prior to use, for example in ayeast-based chromosomal system as described in U.S. Pat. No. 8,563,314.Nuclease expression constructs can be readily designed using methodsknown in the art. Expression of the nuclease may be under the control ofa constitutive promoter or an inducible promoter, for example thegalactokinase promoter which is activated (de-repressed) in the presenceof raffinose and/or galactose and repressed in presence of glucose.

RNA Components of CRISPR/Cas

The Cas9 related CRISPR/Cas system comprises two RNA non-codingcomponents: tracrRNA and a pre-crRNA array containing nuclease guidesequences (spacers) interspaced by identical direct repeats (DRs). Touse a CRISPR/Cas system to accomplish genome engineering, both functionsof these RNAs must be present (see Cong et al, (2013) Sciencexpress1/10.1126/science 1231143). In some embodiments, the tracrRNA andpre-crRNAs are supplied via separate expression constructs or asseparate RNAs. In other embodiments, a chimeric RNA is constructed wherean engineered mature crRNA (conferring target specificity) is fused to atracrRNA (supplying interaction with the Cas9) to create a chimericcr-RNA-tracrRNA hybrid (also termed a single guide RNA). (see Jinek ibidand Cong, ibid).

Chimeric or sgRNAs can be engineered to comprise a sequencecomplementary to any desired target. The RNAs comprise 22 bases ofcomplementarity to a target and of the form G[n19], followed by aprotospacer-adjacent motif (PAM) of the form NGG. Thus, in one method,sgRNAs can be designed by utilization of a known ZFN target in a gene ofinterest by (i) aligning the recognition sequence of the ZFN heterodimerwith the reference sequence of the relevant genome (human, mouse, or ofa particular plant species); (ii) identifying the spacer region betweenthe ZFN half-sites; (iii) identifying the location of the motif G[N20]GGthat is closest to the spacer region (when more than one such motifoverlaps the spacer, the motif that is centered relative to the spaceris chosen); (iv) using that motif as the core of the sgRNA. This methodadvantageously relies on proven nuclease targets. Alternatively, sgRNAscan be designed to target any region of interest simply by identifying asuitable target sequence the conforms to the G[n20]GG formula. Alongwith the complementarity region, an sgRNA may comprise additionalnucleotides to extend to tail region of the tracrRNA portion of thesgRNA (see Hsu et al (2013) Nature Biotech doi:10.1038/nbt.2647). Tailsmay be of +67 to +85 nucleotides, or any number therebetween with apreferred length of +85 nucleotides. Truncated sgRNAs may also be used,“tru-gRNAs” (see Fu et al, (2014) Nature Biotech 32(3): 279). Intru-gRNAs, the complementarity region is diminished to 17 or 18nucleotides in length.

Further, alternative PAM sequences may also be utilized, where a PAMsequence can be NAG as an alternative to NGG (Hsu 2014, ibid) using a S.pyogenes Cas9. Additional PAM sequences may also include those lackingthe initial G (Sander and Joung (2014) Nature Biotech 32(4):347). Inaddition to the S. pyogenes encoded Cas9 PAM sequences, other PAMsequences can be used that are specific for Cas9 proteins from otherbacterial sources. For example, the PAM sequences shown below (adaptedfrom Sander and Joung, ibid, and Esvelt et al, (2013) Nat Meth10(11):1116) are specific for these Cas9 proteins:

Species PAM S. pyogenes NGG S. pyogenes NAG S. mutans NGGS. thermophilius NGGNG S. thermophilius NNAAAW S. thermophilius NNAGAAS. thermophilius NNNGATT C. jejuni NNNNACA N. meningitides NNNNGATTP. multocida GNNNCNNA F. novicida NG

Thus, a suitable target sequence for use with a S. pyogenes CRISPR/Cassystem can be chosen according to the following guideline: [n17, n18,n19, or n20](G/A)G. Alternatively the PAM sequence can follow theguideline G[n17, n18, n19, n20](G/A)G. For Cas9 proteins derived fromnon-S. pyogenes bacteria, the same guidelines may be used where thealternate PAMs are substituted in for the S. pyogenes PAM sequences.

Most preferred is to choose a target sequence with the highestlikelihood of specificity that avoids potential off target sequences.These undesired off target sequences can be identified by consideringthe following attributes: i) similarity in the target sequence that isfollowed by a PAM sequence known to function with the Cas9 protein beingutilized; ii) a similar target sequence with fewer than three mismatchesfrom the desired target sequence; iii) a similar target sequence as inii), where the mismatches are all located in the PAM distal regionrather than the PAM proximal region (there is some evidence thatnucleotides 1-5 immediately adjacent or proximal to the PAM, sometimesreferred to as the ‘seed’ region (Wu et al (2014) Nature Biotechdoi:10.1038/nbt2889) are the most critical for recognition, so putativeoff target sites with mismatches located in the seed region may be theleast likely be recognized by the sg RNA); and iv) a similar targetsequence where the mismatches are not consecutively spaced or are spacedgreater than four nucleotides apart (Hsu 2014, ibid). Thus, byperforming an analysis of the number of potential off target sites in agenome for whichever CRIPSR/Cas system is being employed, using thesecriteria above, a suitable target sequence for the sgRNA may beidentified.

Donors

As noted above, insertion of an exogenous sequence (also called a “donorsequence” or “donor” or “transgene”), for example for correction of amutant gene or for increased expression of a wild-type gene. It will bereadily apparent that the donor sequence is typically not identical tothe genomic sequence where it is placed. A donor sequence can contain anon-homologous sequence flanked by two regions of homology to allow forefficient HDR at the location of interest. Alternatively, a donor mayhave no regions of homology to the targeted location in the DNA and maybe integrated by NHEJ-dependent end joining following cleavage at thetarget site. Additionally, donor sequences can comprise a vectormolecule containing sequences that are not homologous to the region ofinterest in cellular chromatin. A donor molecule can contain several,discontinuous regions of homology to cellular chromatin. For example,for targeted insertion of sequences not normally present in a region ofinterest, said sequences can be present in a donor nucleic acid moleculeand flanked by regions of homology to sequence in the region ofinterest.

The donor polynucleotide can be DNA or RNA, single-stranded and/ordouble-stranded and can be introduced into a cell in linear or circularform. See, e.g., U.S. Patent Publication Nos. 20100047805 and20110207221. If introduced in linear form, the ends of the donorsequence can be protected (e.g., from exonucleolytic degradation) bymethods known to those of skill in the art. For example, one or moredideoxynucleotide residues are added to the 3′ terminus of a linearmolecule and/or self-complementary oligonucleotides are ligated to oneor both ends. See, for example, Chang et al. (1987) Proc. Natl. Acad.Sci. USA84:4959-4963; Nehls et al. (1996) Science 272:886-889.Additional methods for protecting exogenous polynucleotides fromdegradation include, but are not limited to, addition of terminal aminogroup(s) and the use of modified internucleotide linkages such as, forexample, phosphorothioates, phosphoramidates, and O-methyl ribose ordeoxyribose residues.

A polynucleotide can be introduced into a cell as part of a vectormolecule having additional sequences such as, for example, replicationorigins, promoters and genes encoding antibiotic resistance. Moreover,donor polynucleotides can be introduced as naked nucleic acid, asnucleic acid complexed with an agent such as a liposome or poloxamer, orcan be delivered by viruses (e.g., adenovirus, AAV, herpesvirus,retrovirus, lentivirus and integrase defective lentivirus (IDLV)).

The donor is generally inserted so that its expression is driven by theendogenous promoter at the integration site, namely the promoter thatdrives expression of the endogenous gene into which the donor isinserted (e.g., globin, AAVS1, etc.). However, it will be apparent thatthe donor may comprise a promoter and/or enhancer, for example aconstitutive promoter or an inducible or tissue specific promoter.

The donor molecule may be inserted into an endogenous gene such thatall, some or none of the endogenous gene is expressed. In otherembodiments, the transgene (e.g., with or without globin encodingsequences) is integrated into any endogenous locus, for example asafe-harbor locus. See, e.g., US patent publications 20080299580;20080159996 and 201000218264.

Furthermore, although not required for expression, exogenous sequencesmay also include transcriptional or translational regulatory sequences,for example, promoters, enhancers, insulators, internal ribosome entrysites, sequences encoding 2A peptides and/or polyadenylation signals.

Delivery

The nucleases, polynucleotides encoding these nucleases, donorpolynucleotides and compositions comprising the proteins and/orpolynucleotides described herein may be delivered in vivo or ex vivo byany suitable means.

Methods of delivering nucleases as described herein are described, forexample, in U.S. Pat. Nos. 6,453,242; 6,503,717; 6,534,261; 6,599,692;6,607,882; 6,689,558; 6,824,978; 6,933,113; 6,979,539; 7,013,219; and7,163,824, the disclosures of all of which are incorporated by referenceherein in their entireties.

Nucleases and/or donor constructs as described herein may also bedelivered using vectors containing sequences encoding one or more of theCRISPR/Cas system(s). Any vector systems may be used including, but notlimited to, plasmid vectors, DNA minicircles, retroviral vectors,lentiviral vectors, adenovirus vectors, poxvirus vectors; herpesvirusvectors and adeno-associated virus vectors, etc., and combinationsthereof. See, also, U.S. Pat. Nos. 6,534,261; 6,607,882; 6,824,978;6,933,113; 6,979,539; 7,013,219; and 7,163,824, and U.S. patentapplication Ser. No. 14/271,008, incorporated by reference herein intheir entireties. Furthermore, it will be apparent that any of thesevectors may comprise one or more of the sequences needed for treatment.Thus, when one or more nucleases and a donor construct are introducedinto the cell, the nucleases and/or donor polynucleotide may be carriedon the same vector or on different vectors. When multiple vectors areused, each vector may comprise a sequence encoding one or multiplenucleases and/or donor constructs.

Conventional viral and non-viral based gene transfer methods can be usedto introduce nucleic acids encoding nucleases and donor constructs incells (e.g., mammalian cells) and target tissues. Non-viral vectordelivery systems include DNA plasmids, DNA minicircles, naked nucleicacid, and nucleic acid complexed with a delivery vehicle such as aliposome or poloxamer. Viral vector delivery systems include DNA and RNAviruses, which have either episomal or integrated genomes after deliveryto the cell. For a review of gene therapy procedures, see Anderson,Science 256:808-813 (1992); Nabel & Feigner, TIBTECH 11:211-217 (1993);Mitani & Caskey, TIBTECH 11:162-166 (1993); Dillon, TIBTECH 11:167-175(1993); Miller, Nature 357:455-460 (1992); Van Brunt, Biotechnology6(10):1149-1154 (1988); Vigne, Restorative Neurology and Neuroscience8:35-36 (1995); Kremer & Perricaudet, British Medical Bulletin51(1):31-44 (1995); Haddada et al., in Current Topics in Microbiologyand Immunology Doerfler and Böhm (eds.) (1995); and Yu et al., GeneTherapy 1:13-26 (1994).

Methods of non-viral delivery of nucleic acids include electroporation,lipofection, microinjection, biolistics, virosomes, liposomes,immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA,naked RNA, capped RNA, artificial virions, and agent-enhanced uptake ofDNA. Sonoporation using, e.g., the Sonitron 2000 system (Rich-Mar) canalso be used for delivery of nucleic acids.

Additional exemplary nucleic acid delivery systems include thoseprovided by Amaxa Biosystems (Cologne, Germany), Maxcyte, Inc.(Rockville, Md.), BTX Molecular Delivery Systems (Holliston, Mass.) andCopernicus Therapeutics Inc, (see for example U.S. Pat. No. 6,008,336).Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386; 4,946,787;and 4,897,355) and lipofection reagents are sold commercially (e.g.,Transfectam™ and Lipofectin™). Cationic and neutral lipids that aresuitable for efficient receptor-recognition lipofection ofpolynucleotides include those of Feigner, WO 91/17424, WO 91/16024.

The preparation of lipid:nucleic acid complexes, including targetedliposomes such as immunolipid complexes, is well known to one of skillin the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese etal., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem.5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gaoet al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res.52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871,4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).

Additional methods of delivery include the use of packaging the nucleicacids to be delivered into EnGeneIC delivery vehicles (EDVs). These EDVsare specifically delivered to target tissues using bispecific antibodieswhere one arm of the antibody has specificity for the target tissue andthe other has specificity for the EDV. The antibody brings the EDVs tothe target cell surface and then the EDV is brought into the cell byendocytosis. Once in the cell, the contents are released (see MacDiarmidet at (2009) Nature Biotechnology 27(7):643).

The use of RNA or DNA viral based systems for the delivery of nucleicacids encoding engineered CRISPR/Cas systems take advantage of highlyevolved processes for targeting a virus to specific cells in the bodyand trafficking the viral payload to the nucleus. Viral vectors can beadministered directly to subjects (in vivo) or they can be used to treatcells in vitro and the modified cells are administered to subjects (exvivo). Conventional viral based systems for the delivery of CRISPR/Cassystems include, but are not limited to, retroviral, lentivirus,adenoviral, adeno-associated, vaccinia and herpes simplex virus vectorsfor gene transfer. Integration in the host genome is possible with theretrovirus, lentivirus, and adeno-associated virus gene transfermethods, often resulting in long term expression of the insertedtransgene. Additionally, high transduction efficiencies have beenobserved in many different cell types and target tissues.

The tropism of a retrovirus can be altered by incorporating foreignenvelope proteins, expanding the potential target population of targetcells. Lentiviral vectors are retroviral vectors that are able totransduce or infect non-dividing cells and typically produce high viraltiters. Selection of a retroviral gene transfer system depends on thetarget tissue. Retroviral vectors are comprised of cis-acting longterminal repeats with packaging capacity for up to 6-10 kb of foreignsequence. The minimum cis-acting LTRs are sufficient for replication andpackaging of the vectors, which are then used to integrate thetherapeutic gene into the target cell to provide permanent transgeneexpression. Widely used retroviral vectors include those based uponmurine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), SimianImmunodeficiency virus (SIV), human immunodeficiency virus (HIV), andcombinations thereof (see, e.g., Buchscher et al., J. Virol.66:2731-2739 (1992); Johann et al., J. Virol. 66:1635-1640 (1992);Sommerfelt et al., Virol. 176:58-59 (1990); Wilson et al., J. Virol.63:2374-2378 (1989); Miller et al., J. Virol. 65:2220-2224 (1991);PCT/US94/05700).

In applications in which transient expression is preferred, adenoviralbased systems can be used. Adenoviral based vectors are capable of veryhigh transduction efficiency in many cell types and do not require celldivision. With such vectors, high titer and high levels of expressionhave been obtained. This vector can be produced in large quantities in arelatively simple system. Adeno-associated virus (“AAV”) vectors arealso used to transduce cells with target nucleic acids, e.g., in the invitro production of nucleic acids and peptides, and for in vivo and exvivo gene therapy procedures (see, e.g., West et al., Virology 160:38-47(1987); U.S. Pat. No. 4,797,368; WO 93/24641; Kotin, Human Gene Therapy5:793-801 (1994); Muzyczka, J. Clin. Invest. 94:1351 (1994).Construction of recombinant AAV vectors are described in a number ofpublications, including U.S. Pat. No. 5,173,414; Tratschin et al., Mol.Cell. Biol. 5:3251-3260 (1985); Tratschin, et al., Mol. Cell. Biol.4:2072-2081 (1984); Hermonat & Muzyczka, PNAS 81:6466-6470 (1984); andSamulski et al., J. Virol. 63:03822-3828 (1989).

At least six viral vector approaches are currently available for genetransfer in clinical trials, which utilize approaches that involvecomplementation of defective vectors by genes inserted into helper celllines to generate the transducing agent.

pLASN and MFG-S are examples of retroviral vectors that have been usedin clinical trials (Dunbar et al., Blood 85:3048-305 (1995); Kohn etal., Nat. Med. 1:1017-102 (1995); Malech et al., PNAS 94:22 12133-12138(1997)). PA317/pLASN was the first therapeutic vector used in a genetherapy trial. (Blaese et al., Science 270:475-480 (1995)). Transductionefficiencies of 50% or greater have been observed for MFG-S packagedvectors. (Ellem et al., Immunol Immunother. 44(1):10-20 (1997); Dranoffet al., Hum. Gene Ther. 1:111-2 (1997).

Recombinant adeno-associated virus vectors (rAAV) are a promisingalternative gene delivery systems based on the defective andnonpathogenic parvovirus adeno-associated type 2 virus. All vectors arederived from a plasmid that retains only the AAV 145 base pair (bp)inverted terminal repeats flanking the transgene expression cassette.Efficient gene transfer and stable transgene delivery due to integrationinto the genomes of the transduced cell are key features for this vectorsystem. (Wagner et al., Lancet 351:9117 1702-3 (1998), Kearns et al.,Gene Ther. 9:748-55 (1996)). Other AAV serotypes, including AAV1, AAV3,AAV4, AAV5, AAV6, AAV8, AAV9 and AAVrh10, and all variants thereof, canalso be used in accordance with the present invention.

Replication-deficient recombinant adenoviral vectors (Ad) can beproduced at high titer and readily infect a number of different celltypes. Most adenovirus vectors are engineered such that a transgenereplaces the Ad E1a, E1b, and/or E3 genes; subsequently the replicationdefective vector is propagated in human 293 cells that supply deletedgene function in trans. Ad vectors can transduce multiple types oftissues in vivo, including non-dividing, differentiated cells such asthose found in liver, kidney and muscle. Conventional Ad vectors have alarge carrying capacity. An example of the use of an Ad vector in aclinical trial involved polynucleotide therapy for anti-tumorimmunization with intramuscular injection (Sterman et al., Hum. GeneTher. 7:1083-9 (1998)). Additional examples of the use of adenovirusvectors for gene transfer in clinical trials include Rosenecker et al.,Infection 24:1 5-10 (1996); Sterman et al., Hum. Gene Ther. 9:71083-1089 (1998); Welsh et al., Hum. Gene Ther. 2:205-18 (1995); Alvarezet al., Hum. Gene Ther. 5:597-613 (1997); Topf et al., Gene Ther.5:507-513 (1998); Sterman et al., Hum. Gene Ther. 7:1083-1089 (1998).

Packaging cells are used to form virus particles that are capable ofinfecting a host cell. Such cells include 293 cells, which packageadenovirus, and ψ2 cells or PA317 cells, which package retrovirus. Viralvectors used in gene therapy are usually generated by a producer cellline that packages a nucleic acid vector into a viral particle. Thevectors typically contain the minimal viral sequences required forpackaging and subsequent integration into a host (if applicable), otherviral sequences being replaced by an expression cassette encoding theprotein to be expressed. The missing viral functions are supplied intrans by the packaging cell line. For example, AAV vectors used in genetherapy typically only possess inverted terminal repeat (ITR) sequencesfrom the AAV genome which are required for packaging and integrationinto the host genome. Viral DNA is packaged in a cell line, whichcontains a helper plasmid encoding the other AAV genes, namely rep andcap, but lacking ITR sequences. The cell line is also infected withadenovirus as a helper. The helper virus promotes replication of the AAVvector and expression of AAV genes from the helper plasmid. The helperplasmid is not packaged in significant amounts due to a lack of ITRsequences. Contamination with adenovirus can be reduced by, e.g., heattreatment to which adenovirus is more sensitive than AAV.

In many gene therapy applications, it is desirable that the gene therapyvector be delivered with a high degree of specificity to a particulartissue type. Accordingly, a viral vector can be modified to havespecificity for a given cell type by expressing a ligand as a fusionprotein with a viral coat protein on the outer surface of the virus. Theligand is chosen to have affinity for a receptor known to be present onthe cell type of interest. For example, Han et al., Proc. Natl. Acad.Sci. USA 92:9747-9751 (1995), reported that Moloney murine leukemiavirus can be modified to express human heregulin fused to gp70, and therecombinant virus infects certain human breast cancer cells expressinghuman epidermal growth factor receptor. This principle can be extendedto other virus-target cell pairs, in which the target cell expresses areceptor and the virus expresses a fusion protein comprising a ligandfor the cell-surface receptor. For example, filamentous phage can beengineered to display antibody fragments (e.g., FAB or Fv) havingspecific binding affinity for virtually any chosen cellular receptor.Although the above description applies primarily to viral vectors, thesame principles can be applied to nonviral vectors. Such vectors can beengineered to contain specific uptake sequences which favor uptake byspecific target cells.

Gene therapy vectors can be delivered in vivo by administration to anindividual subject, typically by systemic administration (e.g.,intravenous, intraperitoneal, intramuscular, subdermal, or intracranialinfusion) or topical application, as described below. Alternatively,vectors can be delivered to cells ex vivo, such as cells explanted froman individual patient (e.g., lymphocytes, bone marrow aspirates, tissuebiopsy) or universal donor hematopoietic stem cells, followed byreimplantation of the cells into a patient, usually after selection forcells which have incorporated the vector.

Vectors (e.g., retroviruses, adenoviruses, liposomes, etc.) containingnucleases and/or donor constructs can also be administered directly toan organism for transduction of cells in vivo. Alternatively, naked DNAcan be administered. Administration is by any of the routes normallyused for introducing a molecule into ultimate contact with blood ortissue cells including, but not limited to, injection, infusion, topicalapplication and electroporation. Suitable methods of administering suchnucleic acids are available and well known to those of skill in the art,and, although more than one route can be used to administer a particularcomposition, a particular route can often provide a more immediate andmore effective reaction than another route.

Vectors suitable for introduction of polynucleotides described hereininclude non-integrating lentivirus vectors (IDLV). See, for example, Oryet al. (1996) Proc. Natl. Acad. Sci. USA93:11382-11388; Dull et al.(1998) J. Virol. 72: 8463-8471; Zuffery et al. (1998) J. Virol.72:9873-9880; Follenzi et al. (2000) Nature Genetics 25:217-222; U.S.Patent Publication No 2009/054985.

Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of pharmaceutical compositionsavailable, as described below (see, e.g., Remington's PharmaceuticalSciences, 17th ed., 1989).

It will be apparent that the nuclease-encoding sequences and donorconstructs can be delivered using the same or different systems. Forexample, a donor polynucleotide can be carried by a plasmid, while theone or more nucleases can be carried by a AAV vector. Furthermore, thedifferent vectors can be administered by the same or different routes(intramuscular injection, tail vein injection, other intravenousinjection, intraperitoneal administration and/or intramuscularinjection. The vectors can be delivered simultaneously or in anysequential order.

Formulations for both ex vivo and in vivo administrations includesuspensions in liquid or emulsified liquids. The active ingredientsoften are mixed with excipients which are pharmaceutically acceptableand compatible with the active ingredient. Suitable excipients include,for example, water, saline, dextrose, glycerol, ethanol or the like, andcombinations thereof. In addition, the composition may contain minoramounts of auxiliary substances, such as, wetting or emulsifying agents,pH buffering agents, stabilizing agents or other reagents that enhancethe effectiveness of the pharmaceutical composition.

Plant Delivery

As noted above, DNA and/or RNA may be introduced into (e.g., into thegenome of) a desired plant host by a variety of conventional techniques.For reviews of such techniques see, for example, Weissbach & WeissbachMethods for Plant Molecular Biology (1988, Academic Press, N.Y.) SectionVIII, pp. 421-463; and Grierson & Corey, Plant Molecular Biology (1988,2d Ed.), Blackie, London, Ch. 7-9. See, also, U.S. Patent PublicationNos. 20090205083; 20100199389; 20110167521 and 20110189775, incorporatedherein by reference in their entireties.

For example, polynucleotides may be introduced directly into the genomicDNA of the plant cell using techniques such as electroporation andmicroinjection of plant cell protoplasts, or the polynucleotides can beintroduced directly to plant tissue using biolistic methods, such as DNAparticle bombardment (see, e.g., Klein et al. (1987) Nature 327:70-73).Alternatively, the polynucleotide(s) can be introduced into the plantcell via nanoparticle transformation (see, e.g., U.S. Patent PublicationNo. 20090104700, which is incorporated herein by reference in itsentirety). Alternatively, the DNA/RNA constructs may be combined withsuitable T-DNA border/flanking regions and introduced into aconventional Agrobacterium tumefaciens host vector. Agrobacteriumtumefaciens-mediated transformation techniques, including disarming ofoncogenes and the development and use of binary vectors, are welldescribed in the scientific literature. See, for example Horsch et al.(1984) Science 233:496-498, and Fraley et al. (1983) Proc. Nat'l. Acad.Sci. USA 80:4803.

In addition, gene transfer may be achieved using non-Agrobacteriumbacteria or viruses such as Rhizobium sp. NGR234, Sinorhizoboiummeliloti, Mesorhizobium loti, potato virus X, cauliflower mosaic virusand cassaya vein mosaic virus and/or tobacco mosaic virus, See, e.g.,Chung et al. (2006) Trends Plant Sci. 11(1):1-4.

The virulence functions of the Agrobacterium tumefaciens host willdirect the insertion of a T-strand containing the construct and adjacentmarker into the plant cell DNA when the cell is infected by the bacteriausing binary T-DNA vector (Bevan (1984) Nuc. Acid Res. 12:8711-8721) orthe co-cultivation procedure (Horsch et al. (1985) Science227:1229-1231). Generally, the Agrobacterium transformation system isused to engineer dicotyledonous plants (Bevan et al. (1982) Ann. Rev.Genet 16:357-384; Rogers et al. (1986) Methods Enzymol. 118:627-641).The Agrobacterium transformation system may also be used to transform,as well as transfer, polynucleotides to monocotyledonous plants andplant cells. See U.S. Pat. No. 5,591,616; Hernalsteen et al. (1984) EMBOJ 3:3039-3041; Hooykass-Van Slogteren et al. (1984) Nature 311:763-764;Grimsley et al. (1987) Nature 325:1677-179; Boulton et al. (1989) PlantMol. Biol. 12:31-40; and Gould et al. (1991)Plant Physiol. 95:426-434.

Alternative gene transfer and transformation methods include, but arenot limited to, protoplast transformation through calcium-, polyethyleneglycol (PEG)- or electroporation-mediated uptake of naked DNA/RNA (seePaszkowski et al. (1984) EMBO J 3:2717-2722, Potrykus et al. (1985)Molec. Gen. Genet. 199:169-177; Fromm et al. (1985) Proc. Nat. Acad.Sci. USA 82:5824-5828; and Shimamoto (1989) Nature 338:274-276) andelectroporation of plant tissues (D'Halluin et al. (1992) Plant Cell4:1495-1505). Additional methods for plant cell transformation includemicroinjection, silicon carbide (e.g., WHISKERS™) mediated DNA uptake(Kaeppler et al. (1990) Plant Cell Reporter 9:415-418), andmicroprojectile bombardment (see Klein et al. (1988) Proc. Nat. Acad.Sci. USA85:4305-4309; and Gordon-Kamm et al. (1990) Plant Cell2:603-618).

Transformed plant cells which are produced by any of the abovetransformation techniques can be cultured to regenerate a whole plantwhich possesses the transformed genotype and thus the desired phenotype.Such regeneration techniques rely on manipulation of certainphytohormones in a tissue culture growth medium, typically relying on abiocide and/or herbicide marker which has been introduced together withthe desired nucleotide sequences. Plant regeneration from culturedprotoplasts is described in Evans, et al., “Protoplasts Isolation andCulture” in Handbook of Plant Cell Culture, pp. 124-176, MacmillianPublishing Company, New York, 1983; and Binding, Regeneration of Plants,Plant Protoplasts, pp. 21-73, CRC Press, Boca Raton, 1985. Regenerationcan also be obtained from plant callus, explants, organs, pollens,embryos or parts thereof. Such regeneration techniques are describedgenerally in Klee et al. (1987) Ann. Rev. of Plant Phys. 38:467-486.

Nucleic acids introduced into a plant cell can be used to confer desiredtraits on essentially any plant. A wide variety of plants and plant cellsystems may be engineered for the desired physiological and agronomiccharacteristics described herein using the nucleic acid constructs ofthe present disclosure and the various transformation methods mentionedabove. In preferred embodiments, target plants and plant cells forengineering include, but are not limited to, those monocotyledonous anddicotyledonous plants, such as crops including grain crops (e.g., wheat,maize, rice, millet, barley), fruit crops (e.g., tomato, apple, pear,strawberry, orange), forage crops (e.g., alfalfa), root vegetable crops(e.g., carrot, potato, sugar beets, yam), leafy vegetable crops (e.g.,lettuce, spinach); flowering plants (e.g., petunia, rose,chrysanthemum), conifers and pine trees (e.g., pine fir, spruce); plantsused in phytoremediation (e.g., heavy metal accumulating plants); oilcrops (e.g., sunflower, rape seed) and plants used for experimentalpurposes (e.g., Arabidopsis). Thus, the disclosed methods andcompositions have use over a broad range of plants, including, but notlimited to, species from the genera Asparagus, Avena, Brassica, Citrus,Citrullus, Capsicum, Cucurbita, Daucus, Erigeron, Glycine, Gossypium,Hordeum, Lactuca, Lolium, Lycopersicon, Malus, Manihot, Nicotiana,Orychophragmus, Oryza, Persea, Phaseolus, Pisum, Pyrus, Prunus,Raphanus, Secale, Solanum, Sorghum, Triticum, Vitis, Vigna, and Zea.

The introduction of nucleic acids introduced into a plant cell can beused to confer desired traits on essentially any plant. In certainembodiments, the altered MDH expression/function in plant cells resultsin plants having increased amount of fruit yield, increased biomass ofplant (or fruit of the plant), higher content of fruit flesh,concentrated fruit set, larger plants, increased fresh weight, increaseddry weight, increased solids context, higher total weight at harvest,enhanced intensity and/or uniformity of color of the crop, alteredchemical (e.g., oil, fatty acid, carbohydrate, protein) characteristics,etc.

One with skill in the art will recognize that an exogenous sequence canbe transiently incorporated into a plant cell. The introduction of anexogenous polynucleotide sequence can utilize the cell machinery of theplant cell in which the sequence has been introduced. The expression ofan exogenous polynucleotide sequence comprising a ZFN that istransiently incorporated into a plant cell can be assayed by analyzingthe genomic DNA of the target sequence to identify and determine anyindels, inversions, or insertions. These types of rearrangements resultfrom the cleavage of the target site within the genomic DNA sequence,and the subsequent DNA repair. In addition, the expression of anexogenous polynucleotide sequence can be assayed using methods whichallow for the testing of marker gene expression known to those ofordinary skill in the art. Transient expression of marker genes has beenreported using a variety of plants, tissues, and DNA delivery systems.Transient analyses systems include but are not limited to direct genedelivery via electroporation or particle bombardment of tissues in anytransient plant assay using any plant species of interest. Suchtransient systems would include but are not limited to electroporationof protoplasts from a variety of tissue sources or particle bombardmentof specific tissues of interest. The present disclosure encompasses theuse of any transient expression system to evaluate a site specificendonuclease (e.g., ZFN) and to introduce mutations within an MDH targetgene. Examples of plant tissues envisioned to test in transients via anappropriate delivery system would include but are not limited to leafbase tissues, callus, cotyledons, roots, endosperm, embryos, floraltissue, pollen, and epidermal tissue.

One of skill in the art will recognize that an exogenous polynucleotidesequence can be stably incorporated in transgenic plants. Once theexogenous polynucleotide sequence is confirmed to be operable, it can beintroduced into other plants by sexual crossing. Any of a number ofstandard breeding techniques can be used, depending upon the species tobe crossed.

A transformed plant cell, callus, tissue or plant may be identified andisolated by selecting or screening the engineered plant material fortraits encoded by the marker genes present on the transforming DNA. Forinstance, selection can be performed by growing the engineered plantmaterial on media containing an inhibitory amount of the antibiotic orherbicide to which the transforming gene construct confers resistance.Further, transformed plants and plant cells can also be identified byscreening for the activities of any visible marker genes (e.g., theβ-glucuronidase, luciferase, B or C1 genes) that may be present on therecombinant nucleic acid constructs. Such selection and screeningmethodologies are well known to those skilled in the art.

Physical and biochemical methods also may be used to identify plant orplant cell transformants containing stably inserted gene constructs, orplant cell containing target gene altered genomic DNA which results fromthe transient expression of a site-specific endonuclease (e.g., ZFN).These methods include but are not limited to: 1) Southern analysis orPCR amplification for detecting and determining the structure of therecombinant DNA insert; 2) Northern blot, S1 RNase protection,primer-extension or reverse transcriptase-PCR amplification fordetecting and examining RNA transcripts of the gene constructs; 3)enzymatic assays for detecting enzyme or ribozyme activity, where suchgene products are encoded by the gene construct; 4) protein gelelectrophoresis, Western blot techniques, immunoprecipitation, orenzyme-linked immunoassays (ELISA), where the gene construct productsare proteins. Additional techniques, such as in situ hybridization,enzyme staining, and immunostaining, also may be used to detect thepresence or expression of the recombinant construct in specific plantorgans and tissues. The methods for doing all these assays are wellknown to those skilled in the art.

Effects of gene manipulation using the methods disclosed herein can beobserved by, for example, northern blots of the RNA (e.g., mRNA)isolated from the tissues of interest. Typically, if the mRNA is presentor the amount of mRNA has increased, it can be assumed that thecorresponding transgene is being expressed. Other methods of measuringgene and/or encoded polypeptide activity can be used. Different types ofenzymatic assays can be used, depending on the substrate used and themethod of detecting the increase or decrease of a reaction product orby-product. In addition, the levels of polypeptide expressed can bemeasured immunochemically, i.e., ELISA, RIA, EIA and other antibodybased assays well known to those of skill in the art, such as byelectrophoretic detection assays (either with staining or westernblotting). As one non-limiting example, the detection of the AAD-1 andPAT proteins using an ELISA assay is described in U.S. PatentPublication No. 20090093366, which reference is hereby incorporated byreference in its entirety herein. A transgene may be selectivelyexpressed in some tissues of the plant or at some developmental stages,or the transgene may be expressed in substantially all plant tissues,substantially along its entire life cycle. However, any combinatorialexpression mode is also applicable.

The present disclosure also encompasses seeds of the transgenic plantsdescribed above wherein the seed has the transgene or gene construct.The present disclosure further encompasses the progeny, clones, celllines or cells of the transgenic plants described above wherein saidprogeny, clone, cell line or cell has the transgene or gene construct.

Fusion proteins (e.g., ZFNs) and expression vectors encoding fusionproteins can be administered directly to the plant for gene regulation,targeted cleavage, and/or recombination. In certain embodiments, theplant contains multiple paralogous MDH target genes. Thus, one or moredifferent fusion proteins or expression vectors encoding fusion proteinsmay be administered to a plant in order to target one or more of theseparalogous genes in the plant.

Administration of effective amounts is by any of the routes normallyused for introducing fusion proteins into ultimate contact with theplant cell to be treated. The ZFPs are administered in any suitablemanner, preferably with acceptable carriers. Suitable methods ofadministering such modulators are available and well known to those ofskill in the art, and, although more than one route can be used toadminister a particular composition, a particular route can oftenprovide a more immediate and more effective reaction than another route.

Carriers may also be used and are determined in part by the particularcomposition being administered, as well as by the particular method usedto administer the composition. Accordingly, there is a wide variety ofsuitable formulations of carriers that are available.

EXAMPLES Example 1 Designing of sgRNAs

The sequence of the guide RNA (sgRNA or gRNA) for use in genome editingof a given ZFN-specified locus was identified by: (i) aligning therecognition sequence of the ZFN heterodimer with the reference sequenceof the relevant genome (human, mouse, or of a particular plant species);(ii) identifying the spacer region between the ZFN half-sites; (iii)identifying the location of the motif G[N20]GG that is closest to thespacer region (when more than one such motif overlapped the spacer, themotif that is centered relative to the spacer was chosen); (iv) usingthat motif as the core of the gRNA. Table 1 shows a series of sgRNAs foruse with the CRISPR/Cas system. Cells of interest are contacted with thesgRNAs in Table 1 and are engineered.

TABLE 1  sgRNAs designed to target ZFN target sequences Target Seq IDSeq ID gene Target sequence for ZFN NO: Sequence encoding sgRNA  NO:Human β GGGCAGTAACGGCAGACTTCTCCTCAGG 1 GTCTGCCGTTACTGCCCTGTGGG 149globin ″ TGGGGCAAGGTGAACGTGGATGAAGTTG 2 GTCTGCCGTTACTGCCCTGTGGG 149 ″AGAGTCAGGTGCACCATGGTGTCTGTTT 3 GTAACGGCAGACTTCaCCTCAGG 150 ″GTGGAGAAGTCTGCCGTTACTGCCCTGT 4 GTAACGGCAGACTTCaCCTCAGG 150 ″ACAGGAGTCAGGTGCACCATGGTGTCTG 5 GTAACGGCAGACTTCaCCTCAGG 150 ″GAGAAGTCTGCCGTTACTGCCCTGTGGG 6 GTAACGGCAGACTTCaCCTCAGG 150 ″TAACGGCAGACTTCTCCACAGGAGTCAG 7 GTAACGGCAGACTTCaCCTCAGG 150 ″GCCCTGTGGGGCAAGGTGAACGTGGATG 8 GTAACGGCAGACTTCaCCTCAGG 150 ″GGGCAGTAACGGCAGACTTCTCCTCAGG 1 GTAACGGCAGACTTCaCCTCAGG 150 ″TGGGGCAAGGTGAACGTGGATGAAGTTG 2 GTAACGGCAGACTTCaCCTCAGG 150 ″CACAGGGCAGTAACGGCAGACTTCTCCT 9 GTAACGGCAGACTTCaCCTCAGG 150 ″GGCAAGGTGAACGTGGATGAAGTTGGTG 10 GTAACGGCAGACTTCaCCTCAGG 150 HumanATCCCATGGAGAGGTGGCTGGGAAGGAC 11 GCAATATGAATCCCATGGAGAGG 151 BCL11A ″ATATTGCAGACAATAACCCCTTTAACCT 12 GCAATATGAATCCCATGGAGAGG 151 ″CATCCCAGGCGTGGGGATTAGAGCTCCA 13 GCATATTCTGCACTCATCCCAGG 152 ″GTGCAGAATATGCCCCGCAGGGTATTTG 14 GCATATTCTGCACTCATCCCAGG 152 Human KLF1GGGAAGGGGCCCAGGGCGGTCAGTGTGC 15 GGGCCCCTTCCCGGACACACAGG 153 ″ACACACAGGATGACTTCCTCAAGGTGGG 16 GGGCCCCTTCCCGGACACACAGG 153 ″CGCCACCGGGCTCCGGGCCCGAGAAGTT 17 GCAGGTCTGGGGCGCGCCACCGG 154 ″CCCCAGACCTGCGCTCTGGCGCCCAGCG 18 GCAGGTCTGGGGCGCGCCACCGG 154 ″GGCTCGGGGGCCGGGGCTGGAGCCAGGG 19 GGCCCCCGAGCCCAAGGCGCTGG 155 ″AAGGCGCTGGCGCTGCAACCGGTGTACC 20 GGCCCCCGAGCCCAAGGCGCTGG 155 ″TTGCAGCGCCAGCGCCTTGGGCTCGGGG 21 GCGCTGCAACCGGTGTACCCGGG 156 ″CGGTGTACCCGGGGCCCGGCGCCGGCTC 22 GCGCTGCAACCGGTGTACCCGGG 156 Human γTTGCATTGAGATAGTGTGGGGAAGGGGC 23 GCATTGAGATAGTGTGGGGAAGG 157 regulatory ″ATCTGTCTGAAACGGTCCCTGGCTAAAC 24 GCATTGAGATAGTGTGGGGAAGG 157 ″TTTGCATTGAGATAGTGTGGGGAAGGGG 25 GCATTGAGATAGTGTGGGGAAGG 157 ″CTGTCTGAAACGGTCCCTGGCTAAACTC 26 GCATTGAGATAGTGTGGGGAAGG 157 ″TATTTGCATTGAGATAGTGTGGGGAAGG 27 GCATTGAGATAGTGTGGGGAAGG 157 ″CTGTCTGAAACGGTCCCTGGCTAAACTC 26 GCATTGAGATAGTGTGGGGAAGG 157CTTGACAAGGCAAAC 28 GCTATTGGTCAAGGCAAGGCTGG 158 GTCAAGGCAAGGCTG 29GCTATTGGTCAAGGCAAGGCTGG 158 Human CCR5 GATGAGGATGAC 30GTGTTCATCTTTGGTTTTGTGGG 159 ″ GATGAGGATGAC 30 GTGTTCATCTTTGGTTTTGTGGG159 ″ GATGAGGATGAC 30 GTGTTCATCTTTGGTTTTGTGGG 159 ″ GATGAGGATGAC 30GTGTTCATCTTTGGTTTTGTGGG 159 ″ GATGAGGATGAC 30 GTGTTCATCTTTGGTTTTGTGGG159 ″ GATGAGGATGAC 30 GTGTTCATCTTTGGTTTTGTGGG 159 ″ GATGAGGATGAC 30GTGTTCATCTTTGGTTTTGTGGG 159 ″ AAACTGCAAAAG 31 GTGTTCATCTTTGGTTTTGTGGG159 ″ AAACTGCAAAAG 31 GTGTTCATCTTTGGTTTTGTGGG 159 ″ AAACTGCAAAAG 31GTGTTCATCTTTGGTTTTGTGGG 159 ″ AAACTGCAAAAG 31 GTGTTCATCTTTGGTTTTGTGGG159 ″ AAACTGCAAAAG 31 GTGTTCATCTTTGGTTTTGTGGG 159 ″ AAACTGCAAAAG 31GTGTTCATCTTTGGTTTTGTGGG 159 ″ AAACTGCAAAAG 31 GTGTTCATCTTTGGTTTTGTGGG159 ″ GACAAGCAGCGG 32 GGTCCTGCCGCTGCTTGTCATGG 160 ″ CATCTGCTACTCG 33GGTCCTGCCGCTGCTTGTCATGG 160 Human CXCR4 ATGACTTGTGGGTGGTTGTGTTCCAGTT 34GCTTCTACCCCAATGACTTGTGG 161 ″ GGGTAGAAGCGGTCACAGATATATCTGT 35GCTTCTACCCCAATGACTTGTGG 161 ″ AGTCAGAGGCCAAGGAAGCTGTTGGCTG 36GCCTCTGACTGTTGGTGGCGTGG 162 ″ TTGGTGGCGTGGACGATGGCCAGGTAGC 37GCCTCTGACTGTTGGTGGCGTGG 162 ″ CAGTTGATGCCGTGGCAAACTGGTACTT 38GCCGTGGCAAACTGGTACTTTGG 163 ″ CCAGAAGGGAAGCGTGATGACAAAGAGG 39GCCGTGGCAAACTGGTACTTTGG 163 PPP1R12C ACTAGGGACAGGATTG 40GGGGCCACTAGGGACAGGATTGG 164 ″ CCCCACTGTGGGGTGG 41GGGGCCACTAGGGACAGGATTGG 164 PPP1R12C ACTAGGGACAGGATTG 40GTCACCAATCCTGTCCCTAGTGG 165 ″ CCCCACTGTGGGGTGG 41GTCACCAATCCTGTCCCTAGTGG 165 PPP1R12C ACTAGGGACAGGATTG 40GTGGCCCCACTGTGGGGTGGAGG 166 ″ CCCCACTGTGGGGTGG 41GTGGCCCCACTGTGGGGTGGAGG 166 Mouse and ACCCGCAGTCCCAGCGTCGTGGTGAGCC 42GTCGGCATGACGGGACCGGTCGG 167 Human HPRT ″ GCATGACGGGACCGGTCGGCTCGCGGCA 43GTCGGCATGACGGGACCGGTCGG 167 ″ TGATGAAGGAGATGGGAGGCCATCACAT 44GATGTGATGAAGGAGATGGGAGG 168 ″ ATCTCGAGCAAGACGTTCAGTCCTACAG 45GATGTGATGAAGGAGATGGGAGG 168 ″ AAGCACTGAATAGAAATAGTGATAGATC 46GTGCTTTGATGTAATCCAGCAGG 169 ″ ATGTAATCCAGCAGGTCAGCAAAGAATT 47GTGCTTTGATGTAATCCAGCAGG 169 ″ GGCCGGCGCGCGGGCTGACTGCTCAGGA 48GTCGCCATAACGGAGCCGGCCGG 170 ″ GCTCCGTTATGGCGACCCGCAGCCCTGG 49GTCGCCATAACGGAGCCGGCCGG 170 ″ TGCAAAAGGTAGGAAAAGGACCAACCAG 50GTATTGCAAAAGGTAGGAAAAGG 171 ″ ACCCAGATACAAACAATGGATAGAAAAC 51GTATTGCAAAAGGTAGGAAAAGG 171 ″ CTGGGATGAACTCTGGGCAGAATTCACA 52GCATATCTGGGATGAACTCTGGG 172 ″ ATGCAGTCTAAGAATACAGACAGATCAG 53GCATATCTGGGATGAACTCTGGG 172 ″ TGCACAGGGGCTGAAGTTGTCCCACAGG 54GCCTCCTGGCCATGTGCACAGGG 173 ″ TGGCCAGGAGGCTGGTTGCAAACATTTT 55GCCTCCTGGCCATGTGCACAGGG 173 ″ TTGAATGTGATTTGAAAGGTAATTTAGT 56GAAGCTGATGATTTAAGCTTTGG 174 ″ AAGCTGATGATTTAAGCTTTGGCGGTTT 57GAAGCTGATGATTTAAGCTTTGG 174 ″ GTGGGGTAATTGATCCATGTATGCCATT 58GATCAATTACCCCACCTGGGTGG 175 ″ GGGTGGCCAAAGGAACTGCGCGAACCTC 59GATCAATTACCCCACCTGGGTGG 175 ″ ATCAACTGGAGTTGGACTGTAATACCAG 60GATGTCTTTACAGAGACAAGAGG 176 ″ CTTTACAGAGACAAGAGGAATAAAGGAA 61GATGTCTTTACAGAGACAAGAGG 176 Human CCTATCCATTGCACTATGCTTTATTTAA 62GATCAACAGCACAGGTTTTGTGG 177 albumin ″ CCTATCCATTGCACTATGCTTTATTTAA 62GATCAACAGCACAGGTTTTGTGG 177 ″ CCTATCCATTGCACTATGCTTTATTTAA 62GATCAACAGCACAGGTTTTGTGG 177 ″ CCTATCCATTGCACTATGCTTTATTTAA 62GATCAACAGCACAGGTTTTGTGG 177 ″ CCTATCCATTGCACTATGCTTTATTTAA 62GATCAACAGCACAGGTTTTGTGG 177 ″ CCTATCCATTGCACTATGCTTTATTTAA 62GATCAACAGCACAGGTTTTGTGG 177 ″ TTTGGGATAGTTATGAATTCAATCTTCA 63GATCAACAGCACAGGTTTTGTGG 177 ″ TTTGGGATAGTTATGAATTCAATCTTCA 63GATCAACAGCACAGGTTTTGTGG 177 ″ TTTGGGATAGTTATGAATTCAATCTTCA 63GATCAACAGCACAGGTTTTGTGG 177 ″ TTTGGGATAGTTATGAATTCAATCTTCA 63GATCAACAGCACAGGTTTTGTGG 177 ″ CCTGTGCTGTTGATCTCATAAATAGAAC 64GATCAACAGCACAGGTTTTGTGG 177 ″ CCTGTGCTGTTGATCTCATAAATAGAAC 64GATCAACAGCACAGGTTTTGTGG 177 ″ TTGTGGTTTTTAAATAAAGCATAGTGCA 65GATCAACAGCACAGGTTTTGTGG 177 ″ TTGTGGTTTTTAAATAAAGCATAGTGCA 65GATCAACAGCACAGGTTTTGTGG 177 ″ ACCAAGAAGACAGACTAAAATGAAAATA 66GATCAACAGCACAGGTTTTGTGG 177 ″ CTGTTGATAGACACTAAAAGAGTATTAG 67GATCAACAGCACAGGTTTTGTGG 177 Human TGACACAGTACCTGGCACCATAGTTGTA 68GTCAGGGTACTAGGGGTATGGGG 178 Factor IX ″ GTACTAGGGGTATGGGGATAAACCAGAC 69GTCAGGGTACTAGGGGTATGGGG 178 Human LRRK2 GCAAAGATTGCTGACTACGGCATTGCTC 70GTCAGCAATCTTTGCAATGATGG 179 ″ TGATGGCAGCATTGGGATACAGTGTGAA 71GTCAGCAATCTTTGCAATGATGG 179 ″ GCAAAGATTGCTGACTACAGCATTGCTC 72GTCAGCAATCTTTGCAATGATGG 179 Human Htt GGGGCGATGCTGGGGACGGGGACATTAG 73 ?″ ACGCTGCGCCGGCGGAGGCGGGGCCGCG 74 GTCTGGGACGCAAGGCGCCGTGG 180 ″AAGGCGCCGTGGGGGCTGCCGGGACGGG 75 GTCTGGGACGCAAGGCGCCGTGG 180 ″AGTCCCCGGAGGCCTCGGGCCGACTCGC 76 GGAGGCCTCGGGCCGACTCGCGG 181 ″GCGCTCAGCAGGTGGTGACCTTGTGGAC 77 GCCGGTGATATGGGCTTCCTGGG 182 ″ATGGTGGGAGAGACTGTGAGGCGGCAGC 78 GAGACTGTGAGGCGGCAGCTGGG 183 ″ATGGCGCTCAGCAGGTGGTGACCTTGTG 79 GAGACTGTGAGGCGGCAGCTGGG 183 ″TGGGAGAGACTGTGAGGCGGCAGCTGGG 80 GAGACTGTGAGGCGGCAGCTGGG 183 Human RHOGCCAGGTAGTACTGTGGGTACTCGAAGG 81 GGCTCAGCCAGGTAGTACTGTGG 184 ″GAGCCATGGCAGTTCTCCATGCTGGCCG 82 GGCTCAGCCAGGTAGTACTGTGG 184 ″CAGTGGGTTCTTGCCGCAGCAGATGGTG 83 GAACCCACTGGGTGACGATGAGG 185 ″GTGACGATGAGGCCTCTGCTACCGTGTC 84 GAACCCACTGGGTGACGATGAGG 185 ″GGGGAGACAGGGCAAGGCTGGCAGAGAG 85 GCCCTGTCTCCCCCATGTCCAGG 186 ″ATGTCCAGGCTGCTGCCTCGGTCCCATT 86 GCCCTGTCTCCCCCATGTCCAGG 186 CFTRATTAGAAGTGAAGTCTGGAAATAAAACC 87 GGGAGAACTGGAGCCTTCAGAGG 187 ″AGTGATTATGGGAGAACTGGATGTTCACAG 88 GGGAGAACTGGAGCCTTCAGAGG 187TCAGTCCACACGTC ″ CATCATAGGAAACACCAAAGATGATATT 89 GAGGGTAAAATTAAGCACAGTGG188 ″ ATATAGATACAGAAGCGTCATCAAAGCA 90 GAGGGTAAAATTAAGCACAGTGG 188 ″GCTTTGATGACGCTTCTGTATCTATATT 91 GAGGGTAAAATTAAGCACAGTGG 188 ″CCAACTAGAAGAGGTAAGAAACTATGTG 92 GAGGGTAAAATTAAGCACAGTGG 188 ″CCTATGATGAATATAGATACAGAAGCGT 93 GAGGGTAAAATTAAGCACAGTGG 188 ″ACACCAATGATATTTTCTTTAATGGTGC 94 GAGGGTAAAATTAAGCACAGTGG 188 TRACCTATGGACTTCAAGAGCAACAGTGCTGT 95 GAGAATCAAAATCGGTGAATAGG 189 ″CTCATGTCTAGCACAGTTTTGTCTGTGA 96 GAGAATCAAAATCGGTGAATAGG 189 ″GTGCTGTGGCCTGGAGCAACAAATCTGA 97 GAGAATCAAAATCGGTGAATAGG 189 ″TTGCTCTTGAAGTCCATAGACCTCATGT 98 GAGAATCAAAATCGGTGAATAGG 189 ″GCTGTGGCCTGGAGCAACAAATCTGACT 99 GACACCTTCTTCCCCAGCCCAGG 190 ″CTGTTGCTCTTGAAGTCCATAGACCTCA 100 GACACCTTCTTCCCCAGCCCAGG 190 ″CTGTGGCCTGGAGCAACAAATCTGACTT 101 GACACCTTCTTCCCCAGCCCAGG 190 ″CTGACTTTGCATGTGCAAACGCCTTCAA 102 GACACCTTCTTCCCCAGCCCAGG 190 ″TTGTTGCTCCAGGCCACAGCACTGTTGC 103 GACACCTTCTTCCCCAGCCCAGG 190 ″TGAAAGTGGCCGGGTTTAATCTGCTCAT 104 GACACCTTCTTCCCCAGCCCAGG 190 ″AGGAGGATTCGGAACCCAATCACTGACA 105 GATTAAACCCGGCCACTTTCAGG 191 ″GAGGAGGATTCGGAACCCAATCACTGAC 106 GATTAAACCCGGCCACTTTCAGG 191 ″TGAAAGTGGCCGGGTTTAATCTGCTCAT 104 GATTAAACCCGGCCACTTTCAGG 191 TRBCCCGTAGAACTGGACTTGACAGCGGAAGT 107 GCTGTCAAGTCCAGTTCTACGGG 192 ″TCTCGGAGAATGACGAGTGGACCCAGGA 108 GCTGTCAAGTCCAGTTCTACGGG 192 ″TCTCGGAGAATGACGAGTGGACCCAGGA 108 GCTGTCAAGTCCAGTTCTACGGG 192 ″TCTCGGAGAATGACGAGTGGACCCAGGA 108 GCTGTCAAGTCCAGTTCTACGGG 192 ″TCTCGGAGAATGACGAGTGGACCCAGGA 108 GCTGTCAAGTCCAGTTCTACGGG 192 ″CCGTAGAACTGGACTTGACAGCGGAAGT 107 GCTGTCAAGTCCAGTTCTACGGG 192 ″CCGTAGAACTGGACTTGACAGCGGAAGT 107 GCTGTCAAGTCCAGTTCTACGGG 192 ″CCGTAGAACTGGACTTGACAGCGGAAGT 107 GCTGTCAAGTCCAGTTCTACGGG 192 ″CCGTAGAACTGGACTTGACAGCGGAAGT 107 GCTGTCAAGTCCAGTTCTACGGG 192 Human PD1CCAGGGCGCCTGTGGGATCTGCATGCCT 109 GGCGCCCTGGCCAGTCGTCTGGG 193 ″CAGTCGTCTGGGCGGTGCTACAACTGGG 110 GGCGCCCTGGCCAGTCGTCTGGG 193 ″GAACACAGGCACGGCTGAGGGGTCCTCC 111 GTCCACAGAGAACACAGGCACGG 194 ″CTGTGGACTATGGGGAGCTGGATTTCCA 112 GTCCACAGAGAACACAGGCACGG 194 ″CAGTCGTCTGGGCGGTGCT 113 GGCGCCCTGGCCAGTCGTCTGGG 193 HumanACAGTGCTTCGGCAGGCTGACAGCCAGG 114 GCTTCGGCAGGCTGACAGCCAGG 195 CTLA-4 ″ACCCGGACCTCAGTGGCTTTGCCTGGAG 115 GCTTCGGCAGGCTGACAGCCAGG 195 ″ACTACCTGGGCATAGGCAACGGAACCCA 116 GTACCCACCGCCATACTACCTGG 196 ″TGGCGGTGGGTACATGAGCTCCACCTTG 117 GTACCCACCGCCATACTACCTGG 196 HLA Cl1:GTATGGCTGCGACGTGGGGTCGGACGGG 118 GCTGCGACGTGGGGTCGGACGGG 197 HLA A2 ″TTATCTGGATGGTGTGAGAACCTGGCCC 119 GCAGCCATACATTATCTGGATGG 198 ″TCCTCTGGACGGTGTGAGAACCTGGCCC 120 GCAGCCATACATCCTCTGGACGG 199 HLA A3ATGGAGCCGCGGGCGCCGTGGATAGAGC 121 GTGGATAGAGCAGGAGGGGCCGG 200 ″CTGGCTCGCGGCGTCGCTGTCGAACCGC 122 GAGCCAGAGGATGGAGCCGCGGG 201 HLA BTCCAGGAGCTCAGGTCCTCGTTCAGGGC 123 GGACCTGAGCTCCTGGACCGCGG 202 ″CGGCGGACACCGCGGCTCAGATCACCCA 124 GGACCTGAGCTCCTGGACCGCGG 202 ″AGGTGGATGCCCAGGACGAGCTTTGAGG 125 GATGCCCAGGACGAGCTTTGAGG 203 ″AGGGAGCAGAAGCAGCGCAGCAGCGCCA 126 GCGCTGCTTCTGCTCCCTGGAGG 204 ″CTGGAGGTGGATGCCCAGGACGAGCTTT 127 GCGCTGCTTCTGCTCCCTGGAGG 204 ″GAGCAGAAGCAGCGCAGCAGCGCCACCT 128 GCGCTGCTTCTGCTCCCTGGAGG 204 HLA CCCTCAGTTTCATGGGGATTCAAGGGAAC 129 GGGGATTCAAGGGAACACCCTGG 205 ″CCTAGGAGGTCATGGGCATTTGCCATGC 130 GCAAATGCCCATGACCTCCTAGG 206 ″TCGCGGCGTCGCTGTCGAACCGCACGAA 131 GAGCCAGAGGATGGAGCCGCGGG 201 ″CCAAGAGGGGAGCCGCGGGAGCCGTGGG 132 GGCGCCCGCGGCTCCCCTCTTGG 207 HLA cl.II:GAAATAAGGCATACTGGTATTACTAATG 133 GTTCACATCTCCCCCGGGCCTGG 208 DBP2 ″GAGGAGAGCAGGCCGATTACCTGACCCA 134 GTTCACATCTCCCCCGGGCCTGG 208 DRATCTCCCAGGGTGGTTCAGTGGCAGAATT 135 GGAGAATGCGGGGGAAAGAGAGG 209 ″GCGGGGGAAAGAGAGGAGGAGAGAAGGA 136 GGAGAATGCGGGGGAAAGAGAGG 209 TAP1AGAAGGCTGTGGGCTCCTCAGAGAAAAT 137 GCCCACAGCCTTCTGTACTCTGG 210 ″ACTCTGGGGTAGATGGAGAGCAGTACCT 138 GCCCACAGCCTTCTGTACTCTGG 210 TAP2TTGCGGATCCGGGAGCAGCTTTTCTCCT 139 GTTGATTCGAGACATGGTGTAGG 211 ″TTGATTCGAGACATGGTGTAGGTGAAGC 140 GTTGATTCGAGACATGGTGTAGG 211 TapasinCCACAGCCAGAGCCTCAGCAGGAGCCTG 141 GCTCTGGCTGTGGTCGCAAGAGG 212 ″CGCAAGAGGCTGGAGAGGCTGAGGACTG 142 GCTCTGGCTGTGGTCGCAAGAGG 212 ″CTGGATGGGGCTTGGCTGATGGTCAGCA 143 GCAGAACTGCCCGCGGGCCCTGG 213 ″GCCCGCGGGCAGTTCTGCGCGGGGGTCA 144 GCAGAACTGCCCGCGGGCCCTGG 213 CIITAGCTCCCAGGCAGCGGGCGGGAGGCTGGA 145 GCTGCCTGGGAGCCCTACTCGGG 214 ″CTACTCGGGCCATCGGCGGCTGCCTCGG 146 GCTGCCTGGGAGCCCTACTCGGG 214 RFX5TTGATGTCAGGGAAGATCTCTCTGATGA 147 GCCTTCGAGCTTTGATGTCAGGG 215 ″GCTCGAAGGCTTGGTGGCCGGGGCCAGT 148 GCCTTCGAGCTTTGATGTCAGGG 215

Example 2 sgRNAs and Tru-RNAs to Genes of Interest

As described in Example 1, the design of an sgRNA is accomplishedthrough a variety of considerations: (i) aligning the recognitionsequence of the ZFN heterodimer with the reference sequence of therelevant genome (human, mouse, or of a particular plant species); (ii)identifying the spacer region between the ZFN half-sites; (iii)identifying the location of the PAM motif relevant to the Cas9 proteinbeing used (for example: G[N17-20](G/A)G when using the S. pyogenesCas9) that is closest to the spacer region (when more than one suchmotif overlapped the spacer, the motif that is centered relative to thespacer was chosen); (iv) using that motif as the core of the sgRNA.

Shown below in Table 2 are exemplary genes for targeting with aCRISPR/Cas system and an sgRNA of the invention. Also indicated is anaccession number of representative example of a cDNA associated withthese genes.

TABLE 2 Exemplary genes for targeting with a CRISPR/Cas system UCSCGenome Brower Representative Acces- Gene name location sion (cDNA)RefSeq HBB chr11: 5246696-5248301 (NM_000518) BCL11A chr2:60684329-60780633 (NM_022893) KLF1 chr19: 12995237-12998017 (NM_006563)HBG1 chr11: 5269502-5271087 (NM_000559) CCR5 chr3: 46411633-46417697(NM_000579) CXCR4 chr2: 136871919-136873813 (NM_001008540) PPP1R12Cchr19: 55602281-55628968 (NM_017607) HPRT chrX: 133594175-133634698(NM_000194) Mouse HPRT chrX: 52988078-53021660 (NM_013556) (assemblyGRCm38/mm10) ALB chr4: 74269972-74287129 (NM_000477) Factor VIII chrX:154064064-154250998 (NM_000132.3) Factor IX chrX: 138612895-138645617(NM_000133) LRRK2 chr12: 40618813-40763086 (NM_198578) Htt chr4:3076237-3245687 (NM_002111) RHO chr3: 129247482-129254187 (NM_000539)CFTR chr7: 117120017-117308718 (NM_000492) TCRA chr6: 42883727-42893575(NM_001243168) TCRB chr7: 142197572-142198055 L36092.2 PD-1 chr2:242792033-242795132 (NM_005018) CTLA-4 chr2: 204732511-204738683(NM_001037631) HLA-A chr6: 29910247-29912868 (NM_002116) HLA-B chr6:31236526-31239913 NM_005514.6 HLA-C chr6: 31236526-31239125(NM_001243042) HLA-DPA chr6: 33032346-33048555 (NM_033554.3) HLA-DQchr6: 32605183-32611429 (NM_002122) HLA-DRA chr6_ssto_hap7: 3754283-(NM_019111) 3759493 LMP7 chr6_dbb_hap3: 4089872- (X66401) 4093057Tapasin chr6: 33271410-33282164 (NM_172208) RFX5 chr1:151313116-151319769 (NM_001025603) CIITA chr16: 10971055-11002744(NM_000246) TAP1 chr6: 32812986-32821748 (NM_000593) TAP2 chr6:32793187-32806547 (NM_000544) TAPBP chr6: 33267472-33282164 DMD chrX:31137345-33229673 (NM_004006) RFX5 chr1: 151313116-151319769 (NM_000449)B. napus FAD3 See PCT publication JN992612 WO2014/039684 B. napus FAD2See PCT publication JN992609 WO2014/039692 Soybean FAD2 SeeUS20140090116 Zea mays ZP15 See U.S. Pat. No. 8,329,986 GBWI-61522(MaizeCyc) B-ketoacyl ACP See U.S. Pat. No. 8,592,645 synthase II(KASII) Tomato MDH See US 20130326725 AY725474 B. napus EPSPS See U.S.Pat. No. 8,399,218 paralogs C + D Paralog D See U.S. Pat. No. 8,399,218Paralog A + B See U.S. Pat. No. 8,399,218 PPP1R12C chr19:55602840-55624858 (NM_017607) (AAVS1) GR 5: 142646254-142783254(NM_000176) IL2RG chrX: 70327254-70331481 (NM_000206) SFTPB chr2:85884440-85895374 (NM_198843)

Single-guide RNAs are then introduced into cells along withpolynucleotides encoding one or more functional domains, and optionallydonor sequences, for modification of the target gene.

All patents, patent applications and publications mentioned herein arehereby incorporated by reference in their entirety.

Although disclosure has been provided in some detail by way ofillustration and example for the purposes of clarity of understanding,it will be apparent to those skilled in the art that various changes andmodifications can be practiced without departing from the spirit orscope of the disclosure. Accordingly, the foregoing descriptions andexamples should not be construed as limiting.

What is claimed is:
 1. A method of modifying the expression of anendogenous gene in a cell, the method comprising the steps of:administering to the cell a first nucleic acid molecule comprising asingle guide RNA that recognizes a target site in the endogenous geneand a second nucleic acid molecule that encodes a functional domain,wherein the functional domain associates with the single guide RNA onthe target site, thereby modifying the expression of the endogenousgene.
 2. The method of claim 1, wherein the functional domain isselected from the group consisting of a transcriptional activationdomain, a transcriptional repression domain and a nuclease domain. 3.The method of claim 2, wherein the functional domain is a TypeIISrestriction enzyme nuclease domain or a Cas protein.
 4. The method ofclaim 2, wherein the functional domain is a transcriptional activationdomain and expression of the endogenous gene is increased.
 5. The methodof claim 2, wherein the functional domain is a transcriptionalrepression domain and expression of the endogenous gene is inhibited. 6.The method of claim 2, wherein the functional domain is a nuclease andthe endogenous gene is cleaved.
 7. The method of claim 6, where thenuclease domain is comprised by the Cas protein.
 8. The method of claim7, where the Cas protein comprises one more nuclease cleavage domains.9. The method of claim 1, wherein the cell is a mammalian or plant cell.10. The method of claim 9, wherein the mammalian cell is a stem cell.11. The method of claim 1, wherein the endogenous gene is selected fromthe group consisting of a mammalian β globin gene (HBB), a gamma globingene (HBG1), a B-cell lymphoma/leukemia 11A (BCL11A) gene, aKruppel-like factor 1 (KLF1) gene, a CCR5 gene, a CXCR4 gene, a PPP1R12C(AAVS1) gene, an hypoxanthine phosphoribosyltransferase (HPRT) gene, analbumin gene, a Factor VIII gene, a Factor IXgene, a Leucine-rich repeatkinase 2 (LRRK2) gene, a Hungtingin (Htt) gene, a rhodopsin (RHO) gene,a Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene, asurfactant protein B gene (SFTPB), a T-cell receptor alpha (TRAC) gene,a T-cell receptor beta (TRBC) gene, a programmed cell death 1 (PD1)gene, a Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4) gene, an humanleukocyte antigen (HLA) A gene, an HLA B gene, an HLA C gene, an HLA-DPAgene, an HLA-DQ gene, an HLA-DRA gene, a LMP7 gene, a Transporterassociated with Antigen Processing (TAP) 1 gene, a TAP2 gene, a tapasingene (TAPBP), a class II major histocompatibility complex transactivator(CIITA) gene, a dystrophin gene (DMD), a glucocorticoid receptor gene(GR), an IL2RG gene and an RFX5 gene.
 12. The method of claim 1, whereinthe endogenous gene is selected from the group consisting of a plantFAD2 gene, a plant FAD3 gene, a plant ZP15 gene, a plant KASII gene, aplant MDH gene, and a plant EPSPS gene.
 13. A single-guide RNA thatbinds to an endogenous gene selected from the group consisting ofmammalian β globin gene (HBB), a gamma globin gene (HBG1), a B-celllymphoma/leukemia 11A (BCL11A) gene, a Kruppel-like factor 1 (KLF1)gene, a CCR5 gene, a CXCR4 gene, a PPP1R12C (AAVS1) gene, anhypoxanthine phosphoribosyltransferase (HPRT) gene, an albumin gene, aFactor VIII gene, a Factor IXgene, a Leucine-rich repeat kinase 2(LRRK2) gene, a Hungtingin (Htt) gene, a rhodopsin (RHO) gene, a CysticFibrosis Transmembrane Conductance Regulator (CFTR) gene, a surfactantprotein B gene (SFTPB), a T-cell receptor alpha (TRAC) gene, a T-cellreceptor beta (TRBC) gene, a programmed cell death 1 (PD1) gene, aCytotoxic T-Lymphocyte Antigen 4 (CTLA-4) gene, an human leukocyteantigen (HLA) A gene, an HLA B gene, an HLA C gene, an HLA-DPA gene, anHLA-DQ gene, an HLA-DRA gene, a LMP7 gene, a Transporter associated withAntigen Processing (TAP) 1 gene, a TAP2 gene, a tapasin gene (TAPBP), aclass II major histocompatibility complex transactivator (CIITA) gene, adystrophin gene (DMD), a glucocorticoid receptor gene (GR), an IL2RGgene and an RFX5 gene.
 14. A single-guide RNA that binds to anendogenous gene selected from the group consisting of a plant FAD2 gene,a plant FAD3 gene, a plant ZP15 gene, a plant KASII gene, a plant MDHgene, and a plant EPSPS gene.
 15. The single-guide RNA of claim 13,selected from the group consisting of SEQ ID NOs:149-215.